Contents Features Hominin Evolution: 05 An Introduction
07 The Evolution of Tools
Humans and 09 Neanderthals milk? 11 got The Evolution of
Lactase Persistence
Epigenetics and 13 Famines
16 Stress: An Evolutionary Perspective
Research
18 Interview
Professor Harold Dibble Department of Anthropology & Penn Museum
Cardiomyogenesis Utilizing Embryonic Stem Cell 21 Promoting Differentiation In vitro Antibiotic and Antimitotic Properties and Cytotoxicity of Ethnopharmacologically Selected Medicinal Plants from the Dominican Republic RNA Silencing Pathways by Identifying Mutant 31 Elucidating Knockouts within Arabidopsis Thaliana !
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26
Editorial Staff WRITING
EDITORS-IN-CHIEF Sarah Murray Vihang Nakhate
WRITING MANAGERS Natalie Neale Donald Zhang
EDITING
Ishmam Ahmed Tyler Boyce Yixuan Geng Carolyn Lye Karanbir Pahil Mike Zhai Edward Zhao
EDITING MANAGERS Maria Lee Vivek Nimgaonkar
LAYOUT MANAGERS Courtney Connolly Carolyn Lye
BUSINESS MANAGER Claudia Cheung
Coby Basal Rami Ezzibdeh Lucy Li Karanbir Pahil
WEBSITE
Terry Sun Adel Qalieh
LAYOUT
Adel Qalieh
BUSINESS
Brad Lowenstein
FACULTY ADVISORS Dr. M. Krimo Bokreta Dr. Jorge SantiagoAviles
About PennScience
PennScience is a student-run peer-reviewed journal of undergraduate research published by the Science and Technology Wing at the University of Pennsylvania. PennScience presents relevant science features, interviews, and research articles from many disciplines, including biological sciences, chemistry, physics, mathematics, geological sciences, and computer sciences. PennScience is a SAC funded organization. For additional information about the journal, including submission guidelines, visit www.pennscience.org or email us at pennscience@gmail.com.
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LETTER FROM THE EDITORS Dear Readers, We are proud to introduce you to our first issue of the 12th volume of PennScience. In keeping with the strong tradition of our journal, we have dedicated ourselves to improving the quality of the work we publish, both in informing our readers on interesting topics in science and in showcasing research conducted by fellow undergraduates. The theme of this issue, hominin evolution, was inspired by a recent and ongoing wave of fascinating discoveries that continues to change the way we view our species and its archaic origins. From the recovery of the oldest hominin genome yet to the astonishing potential integration of three hominin species into one, recent advances have justifiably prompted a resurgence of intrigue around hominin evolution in the popular imagination. The PennScience staff ventured to examine the theme from various perspectives. Sarah Murray first provides an overview of accepted and contested themes in the field. Donald Zhang traces the crucial development of tools by our ancestors while Coby Basal and Natalie Neale discuss the intricate and yet unclear relationship between Neanderthals and modern humans. From a different level of analysis, Vivek Nimgaonkar explores the evolution of lactase persistence through time while Lucy Li and Karanbir Pahil explore how epigenetic phenomena may be crucial to our evolution in both the short and long terms. Rami Ezzibdeh considers the phenomenon of stress from an evolutionary perspective. Finally, we present an interview with Penn Professor Harold Dibble of the Department of Anthropology and the Penn Museum, wherein we learn more about his groundbreaking research and gain an insider’s perspective on the field. We are also pleased to showcase three excellent research papers written by fellow undergraduates. Sharon Kim studied cardiomyogenesis from a developmental perspective through stem cell differentiation. Jephter Buahen investigated medicinal properties of plants in the Dominican Republic. Finally, Michael Schatz studied RNA silencing pathways in Arabidopsis Thaliana. We would like to thank the groups and individuals who have made PennScience possible. First, we would like to thank all our managers and staff for their dedication to and enthusiasm for the journal. We owe our funding to the Student Activities Council and the Science and Technology Wing, without which we could not publish a high-quality journal. We would also like to thank our faculty advisors, Dr. Bokreta and Dr. Santiago-Aviles, for their continued support. Finally, we would like to thank the Penn faculty who took the time to meet with us and share their insights. Sincerely, Sarah Murray and Vihang Nakhate Co-Editors-in-Chief !
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FEATURES
HOMININ EVOLUTION:
AN INTRODUCTION SARAH MURRAY
H
ominin evolution is one of those subjects where every article is dated by the time it hits the stands. We continually discover new species, merge and differentiate old species, and rework our ideas to fully understand the evolution of hominins and how the species Homo sapiens came to exist1,2. It is an exciting, dynamic field of study consistently challenged by the discovery of new specimens and evidence.
Humans and apes, our closest evolutionary relatives, are collectively called the hominoids3. Hominins are all species of hominoids not including apes, or all species after we diverged in our evolutionary tree from chimpanzees3. The only surviving hominin today is us, Homo sapiens. Hominins are separate from apes both morphologically and locomotively. Today, we very easily see these distinctions between humans and apes, but the differences between apes and early hominins were much more subtle. Many of the morphological differences between apes and hominins came over time as hominins evolved, including the size and shape of the jaw, the orientation of the pelvis and spine, and the overall size of the body4. One of the most important distinctions between hominins and apes, even very early on, is that hominins are primarily bipedal4. The earliest known hominin, distinctly not related to chimpanzees or gorillas, is Sahelanthropus tchadensis, which lived from 7 to 6 million years ago5. While not the last common ancestor between apes and hominins, S. tchadensis is the closest hominin so far discovered to that last common ancestor. Researchers have determined that S. tchadensis was a biped that lived in a likely wooded habitat6. The next oldest hominin is Orrorin tugenesis, which lived from 6.1 to 5.8 million years ago5. O. tugenesis was also a biped, though the upper limb fossils also suggest that it climbed trees as well7. After O. tugenesis, a new genus emerged, Ardipithe-
cus. There are two known species in the genus, Ardipithecus kadaba (5.7 to 5.2 million years ago) and Ardipithecus ramidus (4.5 to 4.2 million years ago)5. This genus lived in woodland areas, was omnivorous, and was both bipedal and tree-climbing, much like O. tugenesis8. 4.1 million years ago, the genus Australopithecus emerged. Australopitheci differed from earlier and later hominins in several ways: they were full bipeds, with reduced tree climbing compared to the earlier hominins; their teeth were more similar to humans than earlier hominins, particularly in the size of the anterior teeth and canines; and the phenotypic differences between males and females, or sexual dimorphism, was much more pronounced than in modern humans due to a massive size difference in the two sexes9. The first species in this genus was Au. anamensis, which lived from 4.1 to 3.9 million years ago5. Au. afarensis lived from 3.9 to 2.9 million years ago and has an impressive fossil history, including the famous Lucy skeleton discovered by Donald Johanson and Maurice Taieb in 197410. Other members of this genus include Au. africanus (3 to 2 million years ago), Au. garhi (2.5 million years ago), and Au. sediba (1.95 to 1.75 million years ago)5. Other species of hominins also lived around the same time as the australopitheci. Kenyanthropus platyops, which means “flat-faced man from Kenya,� lived between 3.5 and 3.2 million years ago, and currently there are no other species associated with that genus5. Paranthropus aethiopicus (2.7 to 2.5 million years ago), Paranthropus robustus (2.0 to 1.2 million years ago), and Paranthropus boisei (2.3 to 1.4 million years ago) are three members of the same genus who also lived at the same time as the australopitheci5. Therefore, although Homo sapiens are the only hominin walking the earth today, we know that within the past several million years there was a diverse range of hominins liv-
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FEATURES
ing at the same time, although not necessarily within the same geographic region. The most modern genus of hominins, and the only genus still extant, is Homo. This genus has several differences from Australopithecus, including a more human-like body, “[a] larger brain, systematic meat eating, decreased sexual dimorphism, stereotypic stone tool technologies, and, possibly, rudimentary language and life history changes”9. Currently there are seven different species within this genus, although recent fossil evidence is challenging some of this differentiation1. The oldest member of the genus is H. rudolfensis (2.5 to 1.8 million years ago), which is differentiated from australopitheci by its large cranial capacity. The next oldest member, H. habilis (2.3 to 1.4 million years ago), is commonly associated with simple stone tools. H. erectus survived from 1.8 million to 50 thousand years ago and lived across Eurasia and Africa, making it the first hominin to have such a wide-spread global expansion5,3. H. heidelbergensis, who lived between 800 and 350 thousand years ago, was very similar to humans today in body proportions and cognitive ability, and is known to have hunted large game5,3. H. floresiensis, who lived between 100,000 and 12,000 years ago, was small in body size but similar in cranial size to modern humans, although there is still debate whether specimen assigned to this species really represent a separate species from H. sapiens5. H. neanderthalensis, who lived between 200 and 28 thousand years ago, is a fascinating hominin you can read more about in the article by Coby Basal and Natalie Neale later in this journal. And, of course, the only surviving species of Homos or hominins is Homo sapiens, who emerged 175,000 years ago and are still going strong today. What is important to remember through this evolutionary history is that just because one hominin predates another hominin does not mean the former is a direct ancestor of the latter. There are some hominins that researchers
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have suggested are direct ancestors and descendants, such as Au. anamensis and Au. afarensis, but we cannot say with certainty that this is the case5. We cannot assume that Au. anamensis and Homo sapiens are directly related evolutionarily. This article is not a list of all the hominins that we evolved from or a genetic lineage. What we have outlined in discussing the different species are all the different hominins that have lived before (and with) us. Hominins are our closest biological relatives, and understanding the different species and their evolutionary adaptions can help us see how hominins as a unit evolved to where we as Homo sapiens are today. References 1.
D. Lordkipanidze, M. S. Ponce de León, A. Margvelashvili, Y. Rak, G. P. Rightmire, A. Vekua, C. P. E. Zollikofer, A Complete Skull from Dmanisi, Georgia, and the Evolutionary Biology of Early Homo. Science 342, 326-331 (2013). 2. T. White, Early Hominids—Diversity or Distortion? Science 299, 1994-7 (2003). 3. Pontzer, H., Overview of Hominin Evolution. Nature Education Knowledge (2012). Web. 4. B. Wood, B. Richmond, Human Evolution: taxonomy and paleobiology. Journal of Anatomy 196, 19-60 (2000). 5. Institute of Human Origins, Becoming Human (2009). Web. www.becominghuman.com. 6. M. Brunet, Two New Mio-Pliocene Hominids Enlighten Charles Darwin’s 1871 Prediction. Philosophical Transactions: Biological Sciences 365, 3315-21 (2010). 7. B. G. Richmond, W. L. Jungers, Orrorin tugenesis Femoral Morphology and the Evolution of Hominin Bipedalism. Science 319, 1662-5 (2008). 8. T. D. White, B. Asfaw, Y. Beyene, Y. Haile-Selassie, C. O. Lovejoy, G. Suwa, G. WoldeGabriel, Ardipithecus ramidus and the Paleobiology of Early Hominids. Science 326, 64, 75-86 (2009). 9. C. Ward, The Evolution of Human Origins. American Anthropologist 105, 77-88 (2003). 10. Smithsonian National Museum of Natural History, AL 288-1 (2013). Web. http://humanorigins.si.edu/evidence/humanfossils/fossils/al-288-1
FEATURES
THE EVOLUTION OF
TOOLS DONALD ZHANG
Introduction
What makes humans different from other animal species? This question is harder to answer than it may seem. Humans and animals share many of the same needs and functions: food, water, sleep, and so on. Even more sophisticated “human” traits, like language and emotion, can also be observed in animals1,2.
The use of tools is one of the most important distinguishing characteristics historically used to differentiate humans from animals. Of course, there is again a very blurry line: humans are not the only species to use tools. However, there is a clear hierarchy in terms of the complexity of tool use: the ability to make tools, as seen in chimpanzees using their teeth to sharpen sticks to make spears, is more advanced than simply using natural objects as tools, as seen in capuchin monkeys using rocks to crack open nuts3,4. As with other traits, humans differ from animals in tool use in a stepwise fashion: so far, humans are the only known species that use tools to create other tools5.
crude tools. But it was not until the emergence of Homo habilis, around 2.3 million years ago, that more advanced tools appeared7. Habilis, as the earliest member of genus Homo, bears even more resemblance to modern humans than Australopithecus. Importantly, Homo habilis had a significantly larger brain than Australopithecus, with a cranial capacity of about 650 mL. This first “industry” of more advanced stone tools, named the Oldowan Industry, is characterized by a mode of production called lithic reduction. A large rock, made of material like flint or obsidian, is hit with a hammerstone made out of cobble, causing chips, or flakes, to fracture off. This can be repeated to generate several flakes, as well as an intentionally shaped core. Both the flakes and the core have sharp edges and were used by hominins for various purposes. It is hypothesized that core tools were used like an axe for woodworking, as well as for the cutting and scraping of meat8. Flake tools could have been used for scraping, puncturing, and carving8. These meat-working techniques would have all been in the context of scavenging, as Homo habilis was not a hunter and in fact was often prey for animals like saber-toothed tigers9.
This gives rise to even more questions. Why do we even have tools in the first place? How did we come to develop our use of tools? The obvious answer is that tools were developed simply to make life easier. However, the relationThe Acheulean Industry ship between tools and their hominin users actually runs Around 1.8 million years ago, Homo erectus emerged. much deeper, as can be seen in this overview of the history Again, we see further progression towards the evolution of of hominin tool evolution. modern humans; height was comparable to modern humans, standing at around five-feet-six inches, and cranial The Oldowan Industry 10 In order to fully appreciate the development of tools, it is capacity was 850 mL . A more sophisticated stone tool inhelpful to look at hominins before they had tools. Evolving in dustry known as the “Acheulean” emerged during the same eastern Africa around four million years ago, Australopithe- time period. The technique of lithic reduction was still emcus was one of the first hominins. They were quite small, ployed, but with much more planning of the final shape of standing at around four feet, and had small brains. Their the tool. In particular, the Acheulean industry is charactercranial capacity, at roughly 500 mL, was about a third that ized by the presence of hand axes, flat tools sharpened on of modern humans6. However, they also had some modern both faces with a distinctive pear shape. Hypothesized uses traits, including the ability to walk on two feet. One of the for this multipurpose tool include animal butchering, diglater species, A. garhi, has even been found associated with ging, and chopping. It has even been suggested that they
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FEATURES could have been used as projectile weapons for hunting allowed us to become who we are. small animals, a behavior not seen in the scavenging Homo habilis11. References Perhaps even more significantly, Homo erectus is the first hominin for which there is conclusive evidence for the use of fire12. As another definitive development in hominin evolution, the control of fire allowed for the practice of cooking13. Cooking makes food not only delicious but also easier to digest. In general, this increase in nutrient uptake allows a more active lifestyle to be sustained.
The Mousterian Industry
Finally, the last major development before the appearance of modern humans was the Neanderthals. Evolving around 250,000 years ago, the Neanderthals were closely related to us. They had very large brains, and at around 1450 mL, their cranial capacity was on par with and in some cases greater than that of modern humans14. The “Mousterian Industry” associated with the Neanderthals retains many characteristics of the Acheulean, including hand axes and flakes, but adds increased complexity 15. Neanderthals created denticulates, flake tools with notches in the edges, probably allowing for greater cutting ability16. Neanderthals also made use of spears, which, coupled with observation of trauma on their skeletons, provides strong evidence that Neanderthals hunted big game, like wooly mammoths17. Like Homo erectus, Neanderthals also controlled fire18.
Conclusion
It is easy to simply state that cranial capacity increased for each successive hominin, but it must be appreciated that the dramatic changes in brain size from Homo habilis to Neanderthals are not at all easy to sustain. In modern humans, the brain accounts for more than 20 percent of the resting metabolic rate, compared to around two to eight percent for most other vertebrates19. Thus, the increase in brain size comes with great energetic costs. It has been suggested that hominins may have compensated by evolving a small digestive tract, another system associated with high energy consumption20. The development of increasingly sophisticated tools allowed hunting to become a bigger part of hominin life. The usage of fire made food easier to digest and allowed hominins to absorb more nutrients. Taken together, the prevalence of high-energy meat in the diet and the increased efficiency of nutrient uptake may have made a large digestive tract unnecessary for later hominins and allowed them to develop the large brains humans retain today. Our special aptitude for using tools helps define our species, but it’s also interesting to see how tools have !
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1. L. M. Herman, D. G. Richards, J.P. Wolz, Comprehension of sentences by bottlenosed dolphins. Cognition 16, 129-219 (1984). 2. E. S. Paul, E. J. Harding, M. Mendl, Measuring emotional processes in animals: the utility of a cognitive approach. Neuroscience & Biobehavioral Reviews 29, 469-491 (2005). 3. J. D. Pruetz, P. Bertolani, Savanna Chimpanzees, Pan troglodytes verus, Hunt with Tools. Current Biology 17, 412-417 (2007). 4. M. Fragaszy, Q. Liu, B. W. Wright, A. Allen, C. W. Brown, Wild Bearded Capuchin Monkeys (Sapajus libidinosus) Strategically Place Nuts in a Stable Position during Nut-Cracking. Plos One 9 (2013). 5. S. Sernaw, M. J. Rogers, J. Quade, P. R. Renne, R. F. Butler, M. Dominguez-Rodrigo, D. Stout, W. S. Hart, T. Pickering, S. W. Simpson, 2.6-Million-year-old stone tools and associated bones from OGS-6 and OGS-7, Gona, Afar, Ethiopia. Journal of Human Evolution 45, 169-177 (2003). 6. Smithsonian National Museum of Natural History. What does it mean to be human? http://humanorigins.si.edu/evidence/humanfossils/species/australopithecus-afarensis (accessed Oct 1, 2013). 7. J. Heinzelin, J. D. Clark, T. W. White, W. Hart, P. Renne, G. WoldeGrabriel, Y. Beyene, E. Vrba, Environment and Behavior of 2.5-Million-Year-Old Bouri Hominids. Science 284, 625-629 (1999). 8. 8. Museum of Anthropology. Oldowan and Acheulean Stone Tools. http://anthromuseum.missouri.edu/minigalleries/handaxes/intro. shtml (accessed Oct 1, 2013). 9. J. Lee-Thorp, J. F. Thackeray, N. Merwe, The hunters and the hunted revisited. Journal of Human Evolution 39, 565-576 (2000). 10. Britannica. Homo erectus. http://www.britannica.com/EBchecked/ topic/270386/Homo-erectus (accessed Oct 27, 2013). 11. Samson, D.R. Stones of Contention: The Acheulean Handaxe Lethal Projectile Controversy. Lithic Technology 2006, 31, 2, 127-135 12. F. Berna, P. Goldberg, L. K. Horwitz, J. Brink, S. Holt, M. Bamford, M. Chazan, Microstratigraphic evidence of in situ fire in the Acheulean strata of Wonderwerk Cave, Northern Cape province, South Africa. PNAS 109, 2012. 13. C. Organ, C. L. Nunn, Z. Machanda, R. W. Wrangham, Phylogenetic rate shift in feeding time during the evolution of Homo. PNAS 108, 14555-14559 (2011). 14. Earlham University. Hominid Evolution. http://bioweb.cs.earlham. edu/9-12/hominid/ (accessed Oct 27, 2013). 15. Britannica. Mousterian Industry. http://www.britannica.com/ EBchecked/topic/395112/Mousterian-industry (accessed Oct 1, 2013). 16. Rosen, S. Lithics After the Stone Age: A Handbook of Stone Tools from the Levant; AltaMira Press: Walnut Creek, 1997 17. Dominguez-Rodrigo, M. Stone Tools and Fossil Bones: Debate in the Archaeology of Human Origins; Cambridge University Press: New York, 2012. pg. 161 18. W. Roebroeks, P. Villa, On the earliest evidence for habitual use of fire in Europe. PNAS 108, 5209-5214 (2011). 19. J. W. Mink, R. J. Blumenschine, D. B. Adams, Ratio of central nervous system to body metabolism in vertebrates. Am. J. Physiol. 241, R203-R212 (1981). 20. R. Wrangham, N. Conklin-Brittain, Cooking as a biological trait. Comp. Biochem. Physiol. A. Mol. Integr. Physiol. 136, 35-46 (2003).
FEATURES
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H
COBY BASAL AND NATALIE NEALE
omo neanderthalensis is an extinct species that belongs to the same genus as humans. Both humans and Neanderthals are believed to have come from a common ancestor hundreds of thousands of years ago, and they are both thought to have originated in Africa and journeyed to Eurasia1,2. Scientists have recently found that humans and Neanderthals share extensive DNA. However, they disagree whether this common DNA is exclusively due to sharing a common ancestor or also a result of interbreeding after migrating from Africa.
Neanderthals were similar to humans in many ways. Physiologically, their average brain size was equal or greater to that of modern humans3. Neanderthals also shared several behaviors with modern humans. For example, they controlled fires and hunted large animals4. There is also evidence that they cooked plants for food and possibly medicinal uses5. Additionally, some scientists even argue that Neanderthals explored things that we would consider strictly â&#x20AC;&#x153;human,â&#x20AC;? like the creation of art and symbolic items. For example, some research suggests that Neanderthals created art dating back at least 40,800 years in the Cave of El Castillo in Spain6. Research at the Max Planck Institute for Evolutionary Anthropology has also suggested that Neanderthals created body ornaments in imitation of neighboring Homo sapiens7.
However, despite being the closest extinct relatives of the human beings, Neanderthals exhibit several important differences8. Physically, they were slightly shorter and more broadly built than modern humans9. Their skulls show specialization that is not seen in any other peoples, including the earliest humans, thus contributing to evidence that Neanderthals were a divergent evolutionary lineage separate from Homo sapiens. These specializations include a longer face, a lack of a chin, a long and low braincase, and a juxtamastoid crest behind the mastoid process10. Behaviorally, researchers have found that Neanderthals may have lacked several aspects of what we consider modern. For example, current excavations are challenging the belief that Neanderthal buried their dead11. Researchers are still trying to answer more questions. To what extent did Neanderthals use art and jewelry? Did Neanderthals have language, and to what extent? Ultimately, the more we learn about the striking similarities between humans and Neanderthals, the more we are pressed to find the differences that led to the survival of one species and the extinction of the other. Some scientists argue that Neanderthals were unlikely to have interbred with humans. In fact, there is dating evidence that suggests humans and Neanderthals never met. Neanderthal remains found in the foothills of the Caucasus Mountains have been analyzed with a precise carbon-dating technique and were discovered to be 10,000 years older
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FEATURES than previously thought, suggesting that the Neanderthals died out before humans arrived in Europe12. Scientists are continuing to re-date other Neanderthal sites in Europe with this advanced carbon-dating technique, and expect that these sites will also prove to contain remains that are more ancient than originally deemed. Such findings would give weight to the argument that humans and Neanderthals could never have interbred. Scientists who support this hypothesis further argue that the common DNA of human beings and Neanderthals could be easily explained without resorting to interbreeding13. The shared DNA between human beings and Neanderthals could be the direct result of sharing a common ancestor. However, non-African humans share one to four percent more of their DNA with Neanderthals than African humans, on average13. This is not supported by the hypothesis that humans and Neanderthals never interbred14. If Africans, non-Africans, and Neanderthals shared a common ancestor, and no interbreeding occurred, then Africans and non-Africans should have equal amounts of Neanderthal DNA. This and other evidence have led other scientists to question the theory that humans and Neanderthals did not interbreed.
sharing a common ancestor. If interbreeding is confirmed, this would reveal something about our true identity, whereas if the DNA similarity is simply due to a common ancestor, this will tell us something about our ancestral origins. Complementary to this research, scientists also continue to study whether Neanderthals had similar culture to that of modern humans, which could provide more insight into our relatedness. References 1. 2. 3. 4. 5.
Neanderthal genome analysis has proved to be powerful evidence that humans and Neanderthals did interbreed after humans left Africa. Scientists were able to sequence the Neanderthal genome in 2010 by analyzing tiny chains of DNA taken from the bones of three different Neanderthals that were found in the 1970s15. It was found that on average about 95 to 99 percent of Neanderthal DNA is identical to human DNA on average15. In addition, comparison of the Neanderthal genome to that of modern day humans shows that Neanderthals shared more genetic variants with present day humans in Eurasia than in sub-Saharan Africa13. For example, it was found that Neanderthals carried versions of the human leukocyte antigen genes, a diverse group of immune genes, that are common in modern humans in Europe and Asia, suggesting interbreeding in these regions17. Overall, the shared genetic information suggests that at some point after human beings left Africa, they reproduced with Neanderthals. Since no Neanderthal DNA is observed among Africans, it appears that Neanderthals rarely — if ever — interbred with Africans. The lack of interbreeding between Neanderthals and Africans seems plausible since Neanderthals migrated from Africa tens of thousands of years prior to humans18.
6.
Current genetic and archaeological research still seek to confirm whether the shared DNA between humans and Neanderthals is the result of interbreeding or solely the result of
17.
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7.
8. 9. 10. 11. 12.
13. 14. 15. 16.
J. Noonan et al., Sequencing and analysis of Neanderthal genomic DNA. Science.314, 1113-1118 (2006). S. Sankararaman, N. Patterson, H. Li, S. Pääbo, D. Reich, The Date of Interbreeding Between Neandertals and Modern Humans. PLOS Genetics.8, e1002947 (2012). A. Santa Luca, A re-examination of presumed Neanderthal fossils. Journal of Human Evolution.7, 619-636 (1978). P. Shipman, Separating “us” from “them”: Neanderthal and modern human behavior. Proceedings of the National Academy of Sciences USA.105, 14241-14242 (2008). K. Hardy, et al., Neanderthal medics? Evidence for food, cooking, and medicinal plants entrapped in dental calculus. Naturwissenschaften.99, 617-626 (2012). A.W.G. Pike, et al., U-Series Dating of Paleolithic Art in 11 Caves in Spain. Science.336, 1409-1413 (2012). J.J. Hublin, et al., Radiocarbon Dates from the Grotte du Renne and Saint Cesaire support a Neandertal origin for the Chatelperronian. Proceedings of the National Academy of Sciences USA.109, 18743-18748 (2012). J. Noonan, Neanderthal genomics and the evolution of modern humans. Genomic Res.20, 547-553 (2010). H. Helmuth, Body height, body mass and surface area of the Neandertals. Zeitschrift für Morphologie und Anthropologie.1-12 (1998). A.P. Santa Luca, A re-examination of presumed Neanderthal fossils. Journal of Human Evolution.7, 619-636 (1978). M. Balter, Did Neanderthals Truly Bury Their Dead? Science.337, 1443-1444 (2012). R. Pinhasi, T.F.G. Higham, L.V. Golovanova, V.B. Doronichev, Revised age of late Neanderthal occupation and the end of the Middle Paleolithic in the northern Caucasus. Proceedings of the National Academy of Sciences.108 8611-8616 (2011). R. Green, et al., A Draft Sequence of the Neanderthal Genome. Science.328, 710-722 (2010). A.G.M. Neves, Extremely Rare Interbreeding Events Can Explain Neanderthal DNA in Living Humans. PLoS One.7, e47076 (2012). J.P. Noonan, Neanderthal genomics and the evolution of modern humans. Genomic Research.20, 547-553 (2010). E. Callaway, Ancient DNA reveals secrets of human history. Nature.476, 136-137 (2011). J.J. Hublin, The earliest modern human colonization of Europe. Proceedings of the National Academy of Sciences.109, 1347113472 (2012).
got milk?
FEATURES
The Evolution of
Lactase Persistence VIVEK NIMGAONKAR
I
t is difficult at times to fully rationalize and imagine hominid evolution. Of course, this is in part because evolution is an intergenerational process. Lifetimes are but tiny units of evolutionary time, meaning that we can never truly watch ourselves evolve or witness any dramatic shifts in the genetic or phenotypic composition of our population. Furthermore, in a world of modern medicine, seemingly boundless technology, and fewer geographic barriers, there are veritable buffers to evolution. However, that humans are products of a gradual evolutionary process is well substantiated by considerable evidence, some of which may be waiting in your next glass of milk. 15,000 years ago, if you were to ask a friend, â&#x20AC;&#x153;Got milk?â&#x20AC;? your friend would not only decline but might also be sickened by the thought. For an adult human of that time, drinking milk meant stomach cramps and severe diarrhea. These difficulties in milk digestion can be traced back to a lack of the enzyme lactase. If produced by cells of the intestinal walls, lactase can allow the body to break down lactose, the complex sugar found in milk.
This is not to say that humans of the time period could not produce lactase. In fact, humans not only possessed the gene for lactase but also produced it abundantly under the age of seven or eight1. Expression of the lactase gene made it possible for children to digest and benefit from nutrient-rich breast milk. However, with maturation from childhood, our ancient forefathers would produce less lactase, and in a population of nomadic hunter-gatherers with limited sources of milk, this loss of lactose tolerance was essentially harmless, with few costs to fitness2. Notably, this loss of lactose tolerance in adulthood is
not just some bygone trait of the past. Today, it is believed that just 35% of the human population possesses lactase persistence, the ability to continue to consume milk after childhood3. 99% of the Chinese population does not carry lactose tolerance into adulthood, and other peoples of Southeast Asia and southern Africa possess similar rates of lactase non-persistence1. In sharp contrast, near universal lactase persistence is seen among northern Europeans, western Africans, and certain populations in the Middle East1. It was first hypothesized in anthropological studies of the 1970â&#x20AC;&#x2122;s that a connection may exist between cultures of dairy drinkers and lactase persistence4,5. The practice of cattle domestication developed between !"##$%&'($)$*+,,-./+,.+$0123,"#$!""
FEATURES around 10,500 years ago in North Africa and the Middle East1. This places the development of cattle domestication and milking practices just before the emergence of lactase persistence, which is believed to have emerged and spread in the last 10,000 years6. Thus, it is clearly conceivable that the advent of cattle domestication may have added a selective pressure to maintain the production of lactase beyond childhood. With milk more readily available for consumption, individuals with an ability to tolerate lactose would be able to benefit from the nutritious elements of milk, providing a survival and reproductive advantage. The possibility of a coevolution with culture in this way is not only highly appealing, but also appears to be supported by recent genetic studies, which find evidence of positive selection7.8. Moreover, study has demonstrated that the correlation between cultures that domesticated cattle and populations with lactase persistence is indeed fairly strong, though a few exceptions like the Dinka and Nuer peoples of Sudan and the Somali population in Ethiopia remain3. Despite the appeal and consistency of supporting evidence, the cultural hypothesis of lactase evolution has a few weaknesses. For instance, the argument of selective pressure, which suggests that the ability to digest milk confers a marked selective advantage, encounters some obstacles. Milk is decidedly a valuable source of key nutrients, and one study has even suggested that milk consumption may have increased fertility by as much as 19%9. However, humans did have the means to separate lactose from milk. Studies of Neolithic pottery have found evidence that people in Poland 7,000 years ago had sieves capable of removing lactose-containing whey from milk curds. Why then would humans have required lactase persistence? The best explanation appears to be that lactase persistence spread during times of famine, disease, or drought, when milk may have provided a safe source of water. The difficulties in explaining the selective pressure for lactase persistence and the rapidity of the traitâ&#x20AC;&#x2122;s evolution in parts of Europe present unresolved questions in the story of lactase. Traditionally, the genetics of lactase persistence have eluded explanation. There is no single mutation in the lactase gene that causes lactase persistence. However, one single nucleotide polymorphism (SNP) found 13.9 kilobases upstream of the lactase gene in the intron of a neighboring gene had long been associated with Europe!" !"##$%&"#%"'()*+#,-'.'/,--'0123
an lactase persistence11. Nevertheless, this SNP is largely unobserved in African populations, which for a long time cast doubt on its significance. An important step forward came with a study in 2007, which exposed additional SNPs in African populations and provided strong evidence for convergent evolution of lactase persistence8. The distinct genetics of lactase persistence in African populations suggest that the trait may have evolved independently across different continents, a testament to the vital benefits it likely conferred to our ancestors. The evolution of lactase persistence is an example not only of convergent evolution but also of converging disciplines. The case of lactase lies at the interface of archeology, anthropology, evolutionary biology, and molecular genetics. It illustrates the evolving power of interdisciplinary approaches, and it may demonstrate the potential of complex, multi-faceted culture-genetics coevolution to shape modern hominid evolution. References 1.
M. Leonardi, P. Gerbault, M.G. Thomas, J. Burger, The evolution of lactase persistence in Europe. A synthesis of archaeological and genetic evidence. International Dairy Journal.22, 88-97 (2012). 2. J. Burger, M. Kirchner, B. Bramanti, W. Haak, M.G. Thomas, Absence of the lactase-persistence-associated allele in early Neolithic Europeans. Proceedings of the National Academy of Sciences.104, 3736-3741 (2007). 3. C. J. Ingram, C. A. Mulcare, Y. Itan, M.G. Thomas, D.M. Swallow, Lactose digestion and the evolutionary genetics of lactase persistence. Human genetics.124, 579-591 (2009). 4. R.D. McCracken, Lactase deficiency: an example of dietary evolution. Current Anthropology.12, 479-517 (1971). 5. F.J. Simoons, Primary adult lactose intolerance and the milking habit: A problem in biologic and cultural interrelations. The American Journal of Digestive Diseases.15, 695710 (1970). 6. Y. Itan, A. Powell, M.A. Beaumont, J. Burger, M.G. Thomas, The origins of lactase persistence in Europe. PLoS computational biology.5, e1000491 (2009). 7. T. Bersaglieri, et al., (2004) Genetic signatures of strong recent positive selection at the lactase gene. The American Journal of Human Genetics.74, 1111-1120 (2004). 8. S. A Tishkoff, et al., (2006). Convergent adaptation of human lactase persistence in Africa and Europe. Nature genetics.39, 31-40 (2006). 9. A. Curry, (2013). The milk revolution. Nature.500, 20-22 (2013). 10. M. Salque, et al., Earliest evidence for cheese making in the sixth millennium bc in northern Europe. Nature.493, 522525 (2013). 11. N.S. Enattah, et al., Identification of a variant associated with adult-type hypolactasia. Nature genetics.30, 233-237
FEATURES
AND FAMINES KARANBIR SINGH PAHIL AND LUCY LI ene expression plays a large role in determining the characteristics of an organism. The study of the factors that control gene expression without directly affecting the genome is termed epigenetics. One factor driving epigenetic phenomena is the addition of methyl (-CH3) groups to the backbone of a DNA strand and to histones (proteins that help wind DNA into the very compact subunits that make up chromosomes)1. These additions can occur due to a variety of reasons, including an organismâ&#x20AC;&#x2122;s environment and random chance2. The nature of epigenetic changes allows them to have profound effects on the biology of a population that cannot be fully explained by natural selection for random mutations3,4. In this way, epigenetics may have played a key role in the evolution of humans, providing a mechanism through which environmental factors could alter human phenotypes within the span of a single generation.
G
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FEATURES DNA methylation, the arrangement of histones in DNA, and histone methylation play a large role in DNA accessibility, thus controlling which parts of the genome are transcribed into RNA and then into proteins (1). While it was previously thought that methylation was removed during zygote formation, new research has found a process through which epigenetic traits are passed on through generations. Through this process, termed hydroxylation, the removal of methyl groups from DNA during zygote formation is prevented by the binding of hydroxide (-OH) groups to said methyl groups5. Therefore, epigenetic traits can be passed down from generation to generation. While the genome changes slowly over time, the epigenome can evolve rapidly in response to environmental factors6,7,8. Consequently, epigenetic changes can also affect the evolution of a species3,4,9. The relationship between an individual’s traits and their ancestors’ diets was first examined in 200210. This study investigated the descendants of three different cohorts, born in 1890, 1905 and 1920, from an isolated municipality in northeast Sweden. As was found using records of food prices and harvests from the time, these cohorts were raised in environments with greatly differing abundances of food. It was found that a male’s diet before puberty had a statistically significant effect on his descendants’ health. Shortages of food during a male’s childhood correlated strongly with decreased chances of premature cardiovascular disease mortality and diabetes mortality in their descendants. This relationship between the nutrition of adolescent males and their descendants’ risk of cardiovascular disease and diabetes has been attributed to epigenetics. A famine cannot only affect the epigenome of the descendants of a population, but also that of the population itself11. For example, in a study conducted in 2008, prenatal exposure to the Dutch Hunger Winter in 1944-1945 caused a decrease in methylation of the insulin-like growth factor 2 (IGF2) gene in adults. This decrease in methylation has persisted for over 60 years11. Additionally, prenatal exposure to the Dutch famine also correlated to increased risks of schizophrenia12. The offspring of people who were born around the time of this famine were also at a higher risk of being born with unusually high levels of fat (known as neonatal adiposity) and poor health7. !" !"##$%&"#%"'()*+#,-'.'/,--'0123
As these two studies show, the nutritional environment of an individual during prenatal and adolescent development has significant effects on the epigenetic traits of the individual and his or her offspring. These changes can have negative health implications, such as increased risk of schizophrenia, cardiovascular disease or diabetes. Through further study in the field of epigenetics, we may be able to better understand the mechanisms through which these changes in the epigenome occur. A more comprehensive understanding of how the human epigenome has evolved over time could provide insight into the origins, causes, and possible epigenetic contributions to complex diseases such as schizophrenia13. References 1. R. Jaenisch, A. Bird, Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat Genet.33, 245-54 (2003). 2. J. P. Issa, CpG-island methylation in aging and cancer. Curr Top Microbiol Immunol.249, 101-18 (2000). 3. E. Jablonka, G. Raz, Transgenerational epigenetic inheritance: prevalence, mechanisms, and implications for the study of heredity and evolution. Q Rev Biol.84, 131-76 (2009). 4. M. W Ho, P. T. Saunders, Beyond neo-Darwinism--an epigenetic approach to evolution. J Theor Biol.78, 573-91 (1979). 5. K. Iqbal, S. G. Jin, G. P. Pfeifer, P. E. Szabo, Reprogramming of the paternal genome upon fertilization involves genomewide oxidation of 5-methylcytosine. Proc Natl Acad Sci U S A.108, 3642-7 (2011). 6. N. A. Youngson, E. Whitelaw, Transgenerational epigenetic effects. Annu Rev Genomics Hum Genet.9, 233-57 (2008). 7. R. C. Painter, C. Osmond, P. Gluckman, M. Hanson, D. I. Phillips, T. J. Roseboom, Transgenerational effects of prenatal exposure to the Dutch famine on neonatal adiposity and health in later life. BJOG.115, 1243-9 (2008). 8. G. Kaati, L. O. Bygren, M. Pembrey, M. Sjostrom, Transgenerational response to nutrition, early life circumstances and longevity. Eur J Hum Genet.15, 784-90 (2007). 9. E. V. A. Jablonka, M. J. Lamb, The Changing Concept of Epigenetics. Annals of the New York Academy of Sciences.981, 82-96 (2002). 10. G. Kaati, L. O. Bygren, S. Edvinsson, Cardiovascular and diabetes mortality determined by nutrition during parents’ and grandparents’ slow growth period. Eur J Hum Genet.10, 682-8 (2002). 11. B. T. Heijmans, et al., Persistent epigenetic differences associated with prenatal exposure to famine in humans. Proc Natl Acad Sci U S A.105, 17046-9 (2008). 12. A. S. Brown, E. S. Susser, Prenatal nutritional deficiency and risk of adult schizophrenia. Schizophr Bull.34, 1054-63 (2008). 13. G. Egger, G. Liang, A. Aparicio, P.A. Jones, Epigenetics in human disease and prospects for epigenetic therapy. Nature.429, 457-63 (2004).
Smart Science with a Heart.
FEATURES
Educating a diverse student body
to become knowledgeable, ethical, skillful, and compassionate physicians and biomedical scientists, dedicated to the care of others and health needs of our society in the Jesuit tradition of Cura Personalis, care of the whole person.
School of Medicine !"##$%&'($)$*+,,-./+,.+$0123,"#$!"#
FEATURES
STRESS:
An Evolutionary Perspective
RAMI EZZIBDEH
I
magine taking an exam and coming across a complicated partial differential equation with functions you do not even recognize. We all know what happens next: a sweaty brow, a heavy breath, a pounding heart, and, of course, that feeling of impending doom. All of these physiological adjustments fall under one broad biological function: stress. Stress is a very normal part of our daily lives, perhaps more normal than we might like. Interestingly, even in a decidedly abnormal event, like being chased by lions in the savannah, we would experience very similar physiological responses. Is that calculus exam really as dangerous as a lion? In order to understand how stress operates in humans, we can examine the role of stress in animals. If an animal senses a predator nearby, the stress response triggers a series of biochemical pathways that have substantial physiological effects. Two important hormones in this response are adrenaline and a steroid hormone called glucocorticoid. An increased level of these hormones in the blood stream leads to elevated heart rates, higher blood pressure, and rapid breathing to enhance delivery of oxygen to the muscles1. Those immediate physiological reactions, however, are also accompanied by the temporary shutdown of most â&#x20AC;&#x2DC;non-essentialâ&#x20AC;&#x2122; biological activities, such as growth, tissue repair, and reproductive processes like sperm production or ovulation. The hormones
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therefore act as a selective filter, magnifying the functions that could aid in escaping the threat while postponing the activities that are not absolutely vital for the time being2. Humans experience very similar physiological and hormonal reactions when stressed, whether from being chased by a lion or from taking a test. The fundamental difference in stress response between humans and other animals is what triggers the stress3. For most other species, the response is only limited to times of actual danger, like being chased by a predator. In humans, however, stress can be activated by purely psychological factors. For example, the projected 30-year mortgage rate imposed by the central bank is usually not an imminent lethal threat, but the stress response we experience is physiologically similar to that if we were running for our lives. So then, what is the problem with stress? In lifethreatening situations, it seems to be a vital survival tool. Recently, however, there has been an increasing focus on the negative health impacts of stress, which include increased susceptibility to depression, more severe progression of cancer, and increased vulnerability of the immune system4. The problem actually has less to do with the nature of the stress and more with the amount and duration of exposure. Prolonged levels of intense stress, otherwise known as chronic stress, are what lead to these health problems. The effects of chronic stress have been
FEATURES
demonstrated to be remarkably similar in both primates and humans. Robert Sapolsky, a neuroendocrinologist at Stanford School of Medicine, studied the relationship between stress and social status in Kenyan olive baboons2. Like in humans, the main stressors for these baboons are psychological: they enjoy plentiful food, few predators, and ample free time. Within a group of baboons, there is a clear social hierarchy: those lower on this hierarchy are often victimized, forced to surrender food and mates to dominant baboons. Dominant baboons can also, without warning, decide to violently take out frustration on a subordinate baboon, even if it is simply an innocent bystander. These factors create a long term, unpredictable, stressful lifestyle for subordinate baboons. Sapolsky found that these baboons had higher occurrences of heart disease and higher blood pressure than their dominant counterparts. These results parallel those of the 10-year Whitehall investigation in London, which studied determinants of health in British civil servants5. These people also work in heavily stratified environment, which each person given a precise rank within the hierarchy. The study found that those of lower status had much higher rates of cardiovascular disease and mortality. This cannot be explained by unequal access to healthcare, as all civil servants in the United Kingdom receive the same health benefits. Even after controlling for risk factors such as obesity and smoking, lower grades of civil servants still had higher relative risk for mortality than higher grades. Thus, it has been suggested that chronic stress could be the differentiating factor explaining this disparity. These detrimental effects can be explained physiologically. On the most basic level, our bodies can take one of two states: active or resting. Biochemical pathways in our bodies are divided into catabolic ones, related to the consumption of energy, and anabolic pathways, related to energy storage and tissue repair. The same theme occurs in our nervous system: the sympathetic system is necessary for arousal, increased blood pressure, and other stimulating effects, while the parasympathetic system inhibits muscle activity and is more concerned with body repair6. Stress, in general, biases the active (sympathetic) state in order to maximize the probability of survival. These responses, normally very temporary, are prolonged during chronic stress. This interferes with the bodyâ&#x20AC;&#x2122;s ability to switch into the
resting phase, thus limiting repair and energy restoration7. This results in problems like muscle damage and suppression of the immune system. The cardiovascular responses, when sustained for long enough, can result in hypertension, causing damage to the heart, blood vessels, and kidneys. The question remains of how stress in humans has evolved. On a molecular level, the evolutionary heritage of stress is clear: the same compounds involved in the human stress response, including adrenocorticotropic hormone (ACTH) and corticosteroids, are found in all vertebrates6. Proteins similar to ACTH are found in amphibians and reptiles, and even in insects and mollusks. This high genetic conservation over such a long period of time indicates how useful this ability to change between active and resting states is for survival. It is less clear, however, how psychologically activated stress, as we saw in baboons and humans, evolved. In fact, this is an actively researched topic in the scientific community, and no definitive conclusions have been reached. One current theory is that psychologically triggered stress is necessary in humans to regulate the complex social interactions and activities that distinguish humans from other animals8. As human psychological stress is very distinctive in the animal kingdom, continued research into this area may reveal more about our development as a species. Resources 1. 2. 3. 4. 5. 6. 7. 8.
G.S. Everly, Jr., J.M. Lating. A Clinical Guide to the Treatment of the Human Stress Response. Springer (2013). National Geographic Special Documentary, Stress: Portrait of a Killer (2008). Web. M. Shwartz, Robert Sapolsky Discusses Physiological Effects of Stress. Stanford Report (2007). Web. S. Cohen, D. Janicki-Deverts, G.E. Miller, Psychological Stress and Disease. The Journal of the American Medical Association 298 14 (2007). M.G. Marmot, G.D. Smith. Health Inequalities Among British Civil Servants: The Whitehall II Study. CCSU/Cabinet Office (2004). Web. R.M. Nesse, E.A. Young, Evolutionary Origins and Functions of the Stress Response. Encylopedia of Stress 2 (2000). B.L. Seaward. Managing Stress: Principles and Strategies for Health and Well-Being (Jones and Bartlett, 5 ed., 2006). A. Badyaey. Role of Stress in Evolution: From Individual Adaptability to Evolutionary Adaptation (Elsevier, University of Arizona, 2003).
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INTERVIEW FEATURES
INTERVIEW WITH
Dr. Harold L. Dibble
Conducted by Natalie Neale How did you personally get interested in this field? It’s always been one of my interests. Stone tools have always been one of my interests since I was a kid. I went to college and didn’t really know what I wanted to do — I was a classics major for awhile, then I was a math major for awhile, and it wasn’t until I was a junior that I took my first course in anthropology. I suddenly realized that’s what I wanted to do. I dropped my math major and took as many courses in anthropology as I could before I graduated, and then just continued on. Even then, my interests changed over time. When I first started, I was interested in early Mesopotamian civilizations and studied that for a year or so, then I became interested in domestication of plants and animals (which took place about 10,000 years ago). Then I started working with a faculty member at Arizona, where I was going to school, who was working on Paleolithic, and I realized that’s exactly what I wanted to do. It had everything I love —fossils, stone tools, you name it. It wasn’t something I really set out in life to do, but I am lucky to have found exactly what I wanted to do early on enough so I could build a career on it.
Dr. Harold L. Dibble is a Professor of Anthropology at Penn and Curator-in-Charge of the European Archaeology Section of University of Pennsylvania Museum of Archaeology and Anthropology. He is an established Paleolithic archaeologist known for developing important archaeological techniques, such as use of total station, and for his theory of scraper reduction. His research interests include the archaeology of the Middle Paleolithic of Western Europe, North Africa, and the Near East.
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Basically, the study of human evolution used to be divided in two main areas: whether you looked at the fossils (physical anthropologists) and whether you looked at the stone tools, animal bones, etc. (archaeologists). The field over the past 25 years has changed, and now the entire field of looking at human evolution is called paleoanthropology. I am one of the founding members of the Paleoanthropology Society (founded about 25 years ago), and I was the editor of their journal called Paleoanthropology. The reason for that term was that we needed a term to bring together not only people looking at fossils and not only the people looking at stone tools and animal bones, but also geologists, dating experts, physicists, molecular geneticists, and all of these people who are contributing to the overall study of human evolution. It becomes this incredibly multidisciplinary field. An even broader term — evolutionary anthropology — is now being used to include
INTERVIEW FEATURES psychologists, primatologists, etc. So again, here we are trying to bring together people from all sorts of different disciplines to get a handle of what [humans] are and what we came from. Can you please explain the total station technique and how this technique advanced spatial recording in your field? The reason why we call it a total station is because it measures two kinds of angles when looking at an object — a horizontal angle and a vertical angle. The total station takes these measurements and then adds an electronic distance meter. The electronic distance meter shoots out a laser beam, which bounces off whatever you are looking at and sends it back to the total station. This allows us to measure the distance, so then trigonometrically, with the two angles and the distance, you can figure out the xyz position of whatever object you are looking at. Archaeologists want to get the precise position of all the objects that they dig, and they used to do this by hand and measuring with meter sticks. This technique just does it all automatically and very quickly and accurately, sending it right to the computer. You worked on writing the software for Global Information Systems (GIS). Can you explain how this works? We started this in the early ‘80’s. From the total station, we have all this xyz data in three dimensions. At the time, there was really no software available for dealing with that, so we wrote our own, which allowed us to see the site and see the objects in three dimensions on the computer. We have spatial data from the total station, which includes both horizontal location and depth, so you get to see the distribution of objects through space and across time. Integrating this data with all our other analytical data, you can draw those things in three-dimensional space to get an idea of how the objects are associated with each other. Can you explain your theory of scraper reduction? Imagine you’ve got a piece that was a part of a bigger rock (a flake). You can do various things with this flake, and by hitting the edge it becomes a scraper. You can strike the edge and produce what is called, “retouch” along the edge of the piece. Looking at where
the retouch is, we can define various types of scrapers. Archaeologists did this, and found about 17 different types of these scrapers. Everyone argued about whether the different types were used by different cultures or for different functions. The debate went on for 50 years or so. What I found was that in fact, they’re not different types of tools. What they are, is just tools that are re-sharpened. First you retouch one edge, and if it gets small enough you throw it away. But if you continue to use it, you retouch the other edge and end up with a double scraper. Think of it as pencils — we are arguing about the long pencil people, medium pencil people, and short pencil people, but the differences are really just about how much the tools have been re-sharpened. What are some current field projects you are working on or directing? I’ve excavated six or seven sites in France, and I’ve done a large survey project (which is walking around looking for sites and analyzing surface material) in Egypt, and also excavated at a site called Smuggler’s Cave in Morocco. In addition to that, I’ve analyzed a lot of collections from Iran and Levant. My current excavation right now in France is at a site called La Ferrassie. This site was excavated at the beginning of the 20th Century, where they found a number of Neanderthal remains. We are going back now to figure out the context of the Neanderthal remains. One of the hot topics is whether the Neanderthal buried their dead. This is the second site we’ve looked at from that point of view of considering Neanderthal burials. Previously, there were only four sites that have been discovered that everyone agrees are Neanderthal burials. One of these was a site called Roc de Marsal. Supposedly it was a burial of an infant Neanderthal. We worked there for several seasons, and eventually determined that it was not a Neanderthal burial but was a child that somehow ended up in a natural pit. At La Ferrassie we went back to investigate early claims. The question was whether the remains were really found in a context that would demonstrate deliberate burial. We are trying to see if we get enough information about the context to get an idea about how the remains arrived there. At Smuggler’s Cave, we found the skull of an eight-yearold child about 110,000 years old, and we are looking for evidence again of deliberate burial. One of the big questions in human evolution is what the Neanderthals were doing — did they have rituals and burials? !"##$%&'($)$*+,,-./+,.+$0123,"#$!"#
INTERVIEW FEATURES What it really comes down to is did they have culture What are the biggest challenges in archeological relike what we have with the moderns (art, religion, lan- search for hominid evolution? guage). This is one of the big research questions that I’ve been working on for 30 years or so. Especially in the time period I’m working in (the past 150,000-200,000 years), what we see is a transition from You are also the director of the Laboratory for the what a lot of people would call more archaic forms into Study of Ancient Technology. What are some of the modern Homo sapiens. While there has been some deresearch projects going on there? bate about where that transition has taken place, the large consensus is that the origins of moderns took What we are really interested in at the moment place in Africa somewhere around 120,000 years ago. is doing highly controlled experiments on making From that time on, you have migration of moderns stone tools. By and large, archaeologists missed see- from Africa into other parts of the Old World, and ing the time when people made stone tools. There eventually the New World. As they were going out, they were a few groups here and there, but basically an encountered other populations of more archaic forms, archaeological interest in stone tools developed af- like Neanderthals, and eventually replaced them. So ter most of the world’s populations stopped using we have this change in biology (we don’t look like Nethem, so it’s very much an alien technology. For a anderthals, we look like moderns), and the question is long time, it was debated if the tools found were even what kind of change in behavior was there? This big made by people or other factors (lightning bolts, question revolves around the development of human etc.). What archaeologists had to do is figure out culture as we know it today. When did our ancestors stone tools and how they are made, and in part they develop something that’s like modern human culture did this by teaching themselves how to make them. (things like language, use of symbols and so forth)? We To do this, you take a piece of flint and hit it with don’t want to define it technologically because there are another rock and produce a flake. That flake has lots of different technologies that people use around super sharp edges, and you could modify that flake the world, but we are all still modern. It comes down to be a projectile point. A lot of people, including to what is modern culture, and how do you find it armyself, taught themselves how to make stone tools, chaeologically? That’s where some of the biggest chalwhich is called flintknapping. The trouble with lenges are. I started working in North Africa (in Egypt that is that you get a feeling for how these things and Morocco) to get a better handle on industries (arare made, but it’s hard to really understand because chaeological assemblages) from an early age to see how there are so many variables going into it (how hard distinct they are from what Neanderthals were doing. you strike the stone, at what angle you strike the Neanderthals do not have a lot of evidence for these stone, what kind of material you use to strike the kinds of modern traits. There were four possible sites stone with, what kind of preparation did the stone of Neanderthal burial, one of which we have disproven, have before you struck it). The flintknappers try to and the question now is what about the others? We see identify the roles that each of these variables play, very limited signs of art or symbols for Neanderthals, but it’s a hugely complex thing. What I decided to whereas in Africa, you get a lot more of such evidence do was to develop a way of making stone tools with very early on. There is still a lot of debate as to what a machine that allows us to control these other vari- constitutes modernity. The challenge is to figure this ables so we can isolate particular ones and see the out. If we look at what our closest relatives are doing effect (for example what happens when you change (chimps, bonobos, etc.) we can consider what kinds of material, or hit it harder or softer). These are the behaviors they have. The research is divided into three things we started looking at in a much more sci- fronts: you’ve got looking at primates and seeing what entific way rather than impressionistic way. We’ve they do as one kind of baseline, looking at modern only been doing this for three or four years, and the archeological assemblages and seeing what they did, results are amazing. It turns a lot of what flintknap- and looking at earlier ones or different ones (like Nepers thought on their heads. When you really iso- anderthals) and drawing a contrast there to try to get late the important variables, it redevelops the way a handle on how we can best recognize these kinds of we approach the analysis of stone tools. developments. !" !"##$%&"#%"'()*+#,-'.'/,--'0123
RESEARCH FEATURES
Promoting Cardiomyogenesis Utilizing Embryonic Stem Cell Differentiation Sharon Kim, University of Pennsylvania Advisor: Michelina Iacovino, Ph.D., Pediatric Molecular Genetics, UCLA - Los Angeles Biomedical Research Institute Manipulation of the Wnt/β-catenin signaling pathway within stage-specific phases of mesodermal development can greatly enhance or inhibit cardiomyogenesis . Thus, in this experiment, we attempt to utilize wnt/β-catenin signaling inhibitors on mesodermal progenitor cells to enrich the amount of cardiac progenitors in dissociated embryonic bodies (EBs). Our goal was to create a monolayer of dissociated EBs sorted specifically for mesodermal cardiomyocytes, which is more efficient to analyze than a multi-layer of different germ line cells, where direct cause-and-effect relationships are unclear. During embryonic stem cell (ESC) differentiation, the mesoderm patterns into derived cell parts, specifically targeted according to the expression of Flk1 and PDGRFα markers. Cells expressing Flk1+ PDGRFα - contribute to blood and endothelial cells, while cells expressing PDGRFα + contribute to both cardiac and muscle cells. By comparing cardiomyocyte differentiation among (i) PDGRFα+ cells treated with wnt inhibitor, (ii) non-treated PDGRFα+ cells without wnt inhibitor, (iii) dissociated embryoid bodies (EBs), and (iv) total embryoid bodies, we analyzed whether our goal of enriching cardiac cells in a monolayer of dissociated EBs was reached. Beating cardiomyocytes were absent from both PDGRFα+ cells and dissociated EBs but present in undissociated EBs. This indicates that cardiac progenitor cells were unable to survive in a dissociated state due to two possible reasons: (1) the negative effect of serum-derived factors and (2) the lack of stroma support. In our second experiment, we aimed to control proper mesodermal patterning by performing a time course analysis of the mesoderm makers. FACS analysis indicated that the greatest amount of cardiomyocyte cells (PDGRFα+) was obtained on day 5.5 with a frequency of 27.5% differentiated cardiomyocytes.
I. Introduction
Figure 1
Multipotent embryonic stem cells (ESC) provide the potential to generate different types of cells that could be utilized for regenerative medicine . Several molecules, proteins, cells, and molecular pathways work together to promote efficient induction of the endoderm, mesoderm, and ectoderm in gastrulation . Efficient manipulation of these key molecules and pathways allows for the generation of different cell types including cardiomyocytes. The study of embryonic development has also given insight on the role of progenitor cells, shedding light on their significance in the development of cardiovascular lineages . Identification and isolation of these cardiovascular progenitor cells can lead to helpful models of heart development and provide deeper understanding of the mechanisms regulating cardiovascular lineage diversification. The wnt/β-catenin signaling pathway exhibits developmental stage-specific, biphasic, and antagonistic effects on cardiomyogenesis and hematopoiesis . Inducing the wnt/β-catenin signaling pathway in the early stage of cardiomyogenesis upregulates cardiomyocyte differentiation while inducing it in the late stage downregulates cardiomyocyte differentiation. Furthermore, BMP (Bone Morphogenic Protein) signaling has been identified as an important role of cardiomyocyte differentiation. Interestingly, activation of wnt/β-catenin signaling during the late stage attenuates cardiomyocyte differentiation by suppressing BMP signaling . Collectively, these observations indicate that
Potential of Postnatal Cardiac Progenitor Cells (CPCs). Cardiac progenitor cells are pre-programmed to differentiate into specific types of heart cells such as endothelial, cardiac muscle, or cardiac conduction cells . Generation of cardiac progenitor cells is advantageous in regenerative medicine targeting heart disease or cardiac infarction.
these signaling pathways play some role in the early stages of ESC differentiation. However, the precise stage at which they interact determine their influence on cardiovascular development.
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RESEARCH FEATURES Figure 2
expression markers. Successfully developing a monolayer of cardiomyocyte cells can be very advantageous in research and drug testing where investigators attempt to accurately characterize cell to cell signaling and communication. Analyzing cause and effect relationships and correlations can be difficult when attempting to study a layer of various cell types. Thus, our stage-specific approach to enrich cardiovascular development using embryonic stem cell differentiation would effectively address several medical issues such as regenerative medicine and the treatment of various heart diseases or cardiac infarction.
2. Materials and Methods EXPERIMENT 1 Figure 2: Wnt-β-catenin Signaling Pathway. (a) The wnt protein is unable to bind to the frizzled receptor due to the sFRP repressor protein. Without the accumulation of β-catenin in the cytoplasm, the targeted gene is unable to be expressed. (b) In the absence of the repressor protein sFRP, the wnt protein successfully binds to the frizzled receptor which then allows for β-catenin to accumulate in the cell’s cytoplasm. β-catenin then translocates into the nucleus acting as a transcription coactivator of transcription fac-
The goal of many investigators is efficient and successful production of cardiac progenitors by manipulating key role-playing components in ESC differentiation. Although diverse methods in promoting cardiomyocyte differentiation have been noted, a universally established protocol is still unknown, and several issues can be noted with current methods. For instance, the routine isolation of hematopoetic stem cells (HSCs) from ESCs in serumstimulated cultures is not efficient, since results heavily depend on the type of serum used. In this research project, we attempt to promote cardiomyogenesis in a monolayer of dissociated EBs by increasing the number of cardiac progenitor cells. To achieve this, we utilized a gain-of-function and loss-of-function approach by manipulating the wnt/β-catenin signaling pathway throughout specific time frames of mesodermal development. We attempt to utilize the effects of wnt/β-catenin signaling on dissociated cells sorted for PDGRFα+, which marks for cardiac and muscle embryonic stem cells. By comparing cardiomyocyte differentiation among (i) PDGRFα+ cells treated with wnt inhibitor, (ii) non-treated PDGRFα+ cells, (iii) dissociated embryoid bodies, and (iv) total intact embryoid bodies, we analyzed whether our goal of enriching cardiomyogenesis was reached. Furthermore, we attempted to control ESC differentiation during mesodermal patterning by performing a time course analysis of the mesoderm !! !"##$%&"#%"'()*+#,-'.'/,--'0123
Layout of Experiment
Culture of ES Cells In this experiment, we utilized the E14G embryonic stem cell line, which was plated on MEF (Mouse embryonic fibroblasts) coated gelatinized plate. The E14G cell line was derived from inducible murine ES cells by targeting the gene rTTa into an Inducible Cassette Exchange (ICE) locus . ES cells were cultured in fresh ES cell media that provides an appropriate environment for cell survival. Harvest of ES Cells into EBs After two days of E14G embryonic stem cell culturing, we harvested our cells by breaking apart the ESC colonies. Dissociated ESC colonies allowed for proper preparation of the hanging drop method, which promotes ESC differentiation into distinct germ layers: ectoderm, mesoderm, and endoderm . For our experiment, we particularly focused on the mesoderm, which is where cardiac
RESEARCH FEATURES and blood development occurs.
Isolation and Dissociation of EBs On day 5 of embryonic body development, we collected the hanging drops and plated total embryonic bodies in one well of a gelatinized well plate. We then took a portion of the total EBs and dissociated and divided them into two categories: (1) non-sorted dissociated EBs and (2) dissociated EBs sorted for PDGFRα+, which marks for cardiac and muscle cells. While a portion of the PDGFRα+ cells were untreated and left as the control, a second portion of sorted cells were treated with IRW1, a protein that inhibits the wnt/β-catenin signaling pathway. Hanging Drop Culture Method of Stem Cells: A lab technique used facilitate proper differentiation of ESCs into embryoid bodies of distinct germ layers: endoderm, mesoderm, and ectoderm .
FACS (Fluorescent Activated Cell Sorting) Analysis
was done consecutively on days 3-6 in order to pinpoint a specific time frame within mesodermal development that would exhibit the maximum amount of cardiomyocyte differentiation. After identifying the precise time window that optimizes the concentration of PDGRFα+ sorted cells, we attempted to sort our cells and subsequently treat our fractionated cardiac progenitor cells with DKK1, a protein that inhibits the Wnt/B-catenin signaling pathway, in order to analyze the presence of beating cells.
3. Results On a gelatinized six well plate, we aliquot (i) total EBs, (ii) dissociated EBs, (iii) dissociated EBs sorted for PDGRFα+, and (iv) dissociated EBs sorted for PDGRFα+ treated with IRW1, an inhibiter of the wnt signaling pathway. Three days after collecting our hanging drop culture, approximately 80-100% of the cells in the well containing the total EBs were cardiomyocyte beating cells. However, in the plate containing dissociated EBs, there were no beating cells initially, although a few formed ten days after culturing the hanging drops. Likewise, in both our dissociated EBs sorted for PDGRFα+ and dissociated EBs sorted for PDGRFα+ treated with IRW1, there were no beating cardiomyocyte cells, although secondary EBs had started to form. Figure 3
Cells were sorted through FACS (Fluorescent Activated Cell Sorting) analysis via the flow cytometry machine. Antibody Flk1 was utilized to mark for mesodermal cells while the antibody PDGRFα+ was used to mark for cardiac and muscle cells. The non-sorted EBs, sorted control EBs, and wnt/βcatenin signaling inhibitor induced EBs were all plated in separate wells of the 6 well tray containing the plated total EBs. Throughout embryonic development, we then analyzed the frequency of cardiomyocyte beating cells in our samples.
EXPERIMENT 2 In our second experiment, we attempted to control embryonic stem cell differentiation by performing a time course analysis of the mesodermal expression markers, Flk1 and PDGRFα+. In accordance with our first experiment, the E14G stem cell line was plated on gelatinized plates and cultured into embryonic bodies through hanging drops. After creating hanging drops, FACS analysis
Figure 3: Cell Sorting. E14G EBs sorted and plated on gelatinized plates. Total EBs were plated in well 1, and dissociated EBs were plated in wells 2, 3, and 4. A portion of dissociated EBs were sorted for PDGRFα+ and plated in well 3. Another portion of cells sorted for PDGRFα+ were treated with IRW1 and plated in well 4. In well 1, the frequency was 80-100% of beating cardiomyocytes in total EBs. However, in wells 2, 3, and 4, no beating cardiomyocyte cells were observed.
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RESEARCH FEATURES
Figure 4
Figure 5 Time Course Analysis of Mesodermal Development
Figure 4: Development of Cardiomyocyte Beating Cells from Dissociated Embryonic Bodies. Diagrams show a snapshot of beating cardiomyocyte cells actively contracting under the microscope.
In our second experiment, we attempted to control embryonic stem cell differentiation by performing a time course analysis of the mesoderm expression markers. Although our initial plan was similar to the layout of our first experiment, we were unable to sort and treat our cells due to the lack of cell count. However, sufficient FACS analyses were derived from the flow cytometry machine, which allowed us to quantify the proliferation of cardiac and muscle progenitors in stage-specific time windows of mesodermal development. Consecutive FACS analyses were done on days 3-6, and our data demonstrates that the optimum time window that exhibits the maximum amount of cardiomyocyte differentiation is on day 5.5. The frequency on day 5.5 is 27.5%, which is the maximum amount of cardiomyocyte differentiation among the frequencies exhibited on days 3 - 6.
4. Discussion and Conclusion Judging from the effects of wnt/β-catenin signaling in stage-specific phases in cardiomyogenesis, we hypothesized that an increase in cardiomyocyte differentiation would occur in cells treated with Wnt inhibitors in the late stage of mesodermal development. We also expected to see an enrichment of cardiomyocyte beating cells in dissociated EBs sorted for PDGRFα+, since these have a higher concentration of cardiac and muscle progenitors. In addition, treating dissociated EBs sorted for PDGRFα+ with a wnt inhibitor would hypothetically exhibit the greatest amount of cardiomyocyte differentiation. However, our results demonstrate the absence of beating cells in (i) dissociated embryonic bodies (ii) dissociated EBs sorted for PDGFRα+, and (iii) dissociated EBs sorted for PDGFRα+ treated with wnt inhibitor. Because the dissociated cells sorted for PDGFRα+ were derived from the total EBs, which exhibited a sufficient amount of beating cells, we expected that there would be cardiomyocyte beating in dissociated EBs as well. The lack thereof, however, indicates that the cardiac progenitor cells were not able to survive in a fractionated state due to two possible rea!" !"##$%&"#%"'()*+#,-'.'/,--'0123
Figure 5: Time Course Analysis of Mesodermal Development. FACS (Fluorescent Activated Cell Sorting) Analyses of mesodermal cells on days 3-6 after culturing EB hanging drops. Graphs show that the highest amount of cardiomyocyte cells differentiation is exhibited on day 5 with a frequency of 27.3% differentiated cardiomyocytes.
sons: (1) the negative effects of serum-derived factors and (2) the lack of necessary stroma support in dissociated Ebs. In order to address the first issue, future experiments should be performed under a controlled environment with serumfree media, which would eliminate any inhibitory effects on ESC differentiation. Inserting supplemental cytokines while inhibiting the wnt signaling pathway and activating the BMP signaling pathway can also support successful mesodermal development . In regards to the second issue, dissociated embryoid bodies are likely to have disrupted stroma support thus making the fractionated cells more vulnerable in contrast to total embryoid bodies, which have a protective layer of stroma. Future work should introduce OP9 stroma cells as an alternative to gelatin in order to increase stroma support and ensure cell survival. If stroma cells are not introduced, a potential alternative is to provide extracellular support by introducing matrigel, fibronectin, fibrinogen, or collagen, which allow for the cells to grow in an appropriate
RESEARCH FEATURES environment.
3(1), 55-68.
In our second experiment, we attempted to increase the concentration of PDGFRÎą+ sorted cells that commit to cardiomyocyte cells, subsequently treating the cells with a wnt inhibitor. Although we did not have sufficient number of cells to proceed with sorting, we successfully controlled ESC differentiation by performing a time course analysis of the mesoderm expression markers. Our results show that cardiac and muscle progenitor cells proliferated the most on day 5.5 as demonstrated in Figure 5. Hence, in order to maximize the concentration of PDGRFÎą+ sorted cells, future work should aim to sort and treat cells upon a precise time window of mesodermal development, approximately on day 5.5.
3
In our experiment, we attempted to promote cardiomyogenesis in a monolayer of dissociated EBs sorted specifically for cardiac progenitors. Plating dissociated EBs sorted for specific cell types can produce a monolayer of cells. On the other hand, plating total EBs produces a multi-layer of various cell types from all three germ layers: endoderm, mesoderm, and ectoderm. However, accurately analyzing cell-to-cell signaling or direct causeand-effect relationships is easier in a monolayer of cells as opposed to a multi-layer of various cell types. Thus, promoting cardiomyogenesis among dissociated EBs sorted for specific cell types has much potential in future scientific studies as well as, for example, drug testing where investigators attempt to analyze the effects of a drug on a specific type of cell.
5. Acknowledgements I would like to thank my mentors, Dr. Michelina Iacovino and Dr. Valentina Sanghez, and members of the UCLA Pediatric Molecular Genetics Department for compassionately helping me with my project. I would also like to thank Harbor-UCLA Medical Center and Los Angeles Biomedical Research Institute. Lastly, special thanks to Helen Kim and Raymond Kim who encouraged and inspired me throughout this fellowship. This work was supported by the LA Biomed Summer Fellowship Program for high school graduates class of 2013.
6. References Naito, A. T., Shiojima, I., Akazawa, H., Hidaka, K., Morisaki, T., Kikuchi, A., & Komuro, I. (2006, December 26). Developmental Stage-Specific Biphasic Roles of Wnt/B-Catenin Signaling in Cardiomyogenesis and Hematopoiesis. Proceedings of the National Academy of Sciences, 103(52), 19812-19817.
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Lindsley, R. C., Gill, J. G., Murphy, T. L., Langer, E. M., Cai, M., Mashayekhi, M., . . . Murphy, K. M. (2008, July 3). Mesp Coordinately Regulates Cardiovascular Fate Restriction and EpithelialMesenchymal Transition in Differentiating ESCs. Cell Stem Cell,
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Kattman, S. J., Adler, E. D., & Keller, G. M. (2007, October). Specification of multipotential cardiovascular progenitor cells during embryonic stem cell differentiation and embryonic development. Trends Cardiovasc Med., 17(7), 240-246. Chan, S. S.-K., Shi, X., Toyama, A., Arpke, R. W., Dandapat, A., Iacovino, M., . . . Kyba, M. (2013, May 2). Mesp1 Patterns Mesoderm into Cardiac, Hematopoietic, or Skeletal Myogenic Progenitors in a Context-dependent Manner. Cell Stem Cell, 12(5), 587-601.
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Murry, C. E., & Keller, G. (2008, February 22). Differentiation of Embryonic Stem Cells to Clinically Relevant Populations: Lessons from Embryonic Development. Cell, 132(4), 661-680.
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Ivey, K. N., & Srivastava, D. (2006, June 29). Potential of StemCell-Based Therapies for Heart Disease. Nature, 441, 1097-1099. Retrieved from http://www.nature.com/nature/journal/v441/ n7097/fig_tab/nature04961_F2.html
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Moon, R. T., Kohn, A. D., De Ferrari, G. V., & Kaykas, A. (2005, September). Wnt Beta-Catenin Signaling. Nature Reviews Genetics, 5, 691-701. Retrieved from http://www.nature.com/nrg/journal/v5/n9/fig_tab/nrg1427_F3.html
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Naito, A. T., Shiojima, I., Akazawa, H., Hidaka, K., Morisaki, T., Kikuchi, A., & Komuro, I. (2006, December 26). Developmental Stage-Specific Biphasic Roles of Wnt/B-Catenin Signaling in Cardiomyogenesis and Hematopoiesis. Proceedings of the National Academy of Sciences, 103(52), 19812-19817.
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Naito, A. T., Shiojima, I., Akazawa, H., Hidaka, K., Morisaki, T., Kikuchi, A., & Komuro, I. (2006, December 26). Developmental Stage-Specific Biphasic Roles of Wnt/B-Catenin Signaling in Cardiomyogenesis and Hematopoiesis. Proceedings of the National Academy of Sciences, 103(52), 19812-19817.
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Naito, A. T., Shiojima, I., Akazawa, H., Hidaka, K., Morisaki, T., Kikuchi, A., & Komuro, I. (2006, December 26). Developmental Stage-Specific Biphasic Roles of Wnt/B-Catenin Signaling in Cardiomyogenesis and Hematopoiesis. Proceedings of the National Academy of Sciences, 103(52), 19812-19817. 10
Iacovino, M., Roth, M. E., & Kyba, M. (2011, October). Rapid Genetic Modification of Mouse Embyronic Stem Cells By Inducible Cassette Exchange Recombination. Stem Cells, 29(10), 1580-1588. 11
12 Iacovino, M., Roth, M. E., & Kyba, M. (2011, October). Rapid Genetic Modification of Mouse Embyronic Stem Cells By Inducible Cassette Exchange Recombination. Stem Cells, 29(10), 1580-1588.
Iacovino, M., Roth, M. E., & Kyba, M. (2011, October). Rapid Genetic Modification of Mouse Embyronic Stem Cells By Inducible Cassette Exchange Recombination. Stem Cells, 29(10), 1580-1588. 13
Naito, A. T., Shiojima, I., Akazawa, H., Hidaka, K., Morisaki, T., Kikuchi, A., & Komuro, I. (2006, December 26). Developmental Stage-Specific Biphasic Roles of Wnt/B-Catenin Signaling in Cardiomyogenesis and Hematopoiesis. Proceedings of the National Academy of Sciences, 103(52), 1981219817.
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RESEARCH FEATURES
In vitro Antibiotic and Antimitotic Properties and Cytotoxicity of Ethnopharmacologically Selected Medicinal Plants from the Dominican Republic Jephter Buahen 1a; Anne Osano 1b; Maria Laux 2a, Manuel Aregullin 2b 1 Bowie State University; 2 Cornell University People living in tropical areas such as the Dominican Republic use a broad array of plants for the treatment of diseases such as infections, malaria, diabetes and skin disorders. Studies have shown that many of these plant species possess bioactive properties and can be used to combat health problems. Therefore, this study investigates the antimicrobial, antimitotic and cytotoxic activities of six ethno-pharmacologically selected plants from the Dominican Republic. These plants include Momordica charantia L., Chrysophyllum cainito, Piper marginatum, Lepianthes peltatum, Piper aduncum and Pimenta racemosa. Crude extracts were obtained from the leaves of the selected plants by using two organic solvents; methanol and dichloromethanol. These extracts were screened for their antimicrobial, antimitotic and cytotoxic activities using disc diffusion assay, brine shrimp assay and sea urchin assay. In the disc diffusion assay, strong inhibition against the test microbes was seen for the methanol extract from Momordica charantia. All the crude extracts showed some growth inhibition against the test microbes indicating these plants have antimicrobial properties. The cytotoxicity test of the crude extracts using Brine shrimp also showed the anti-cancer potential of these plants. All the plants extracts were toxic against the Brine shrimp with different efficacies. The Sea Urchin embryo Assay (antimitotic test) showed that all the plants had anti-proliferative properties suggesting their potential ability to stop cancer cell division. Based on the screening of these plants, further studies should focus on isolation of the specific phytochemical constituent of the plants that are responsible for their bioactive properties seen in these exploratory studies.
1. Introduction There are roughly 250,000 higher plant species (angiosperms and gymnosperms) on this planet2. Of these, only about 6% have been screened for biological activity and 15% have undergone phytochemical analysis12. The body of existing ethnomedical knowledge has led to great developments in health care. A report by the WHO Traditional Medicine Programme (TRM) on the evidence that ethnomedical information did indeed lead to useful drug discovery identified a total of 122 compounds. 80% of these compounds were used for the same (or related) ethno medical purposes . Further, it was discovered that these compounds were derived from only 94 species of plants5. The large number of compounds derived from only 94 species of plants, along with the large diversity of flowering plants, means that there should be an abundance of drugs remaining to be discovered in plants. What is the best approach to discover plants that contain potential drugs based on the long-term use of plants by humans? One might expect any bioactive compounds obtained from such plants to have low human toxicity. Obviously, some of these plants may be toxic within a given endemic culture that has no reporting system to document these effects. It is unlikely, however, that acute toxic effects following the use of a plant in these cultures would not be noticed, and the plant would then be used cautiously or not at all. Chronic toxic effects would be less likely to signal that the plant should not be used. In addition, chemical diversity of secondary plant metabolites that result from plant evolution may be equal or superior to that found in synthetic combinatorial chemical libraries. Dominican Republic, just like any other country in the !" !"##$%&"#%"'()*+#,-'.'/,--'0123
tropical areas of the world is very diverse in plants species. This is mainly due to the tropical climate in the region. This has led to many different plant species in a given area. Several plants in the Dominican Republic are used for the treatment of diseases like diabetes, stomach ache, pneumonia, headache etc. The use of these local herbal medicines has been a part of the culture of the local people. The plants selected for this study include: Momoridica charantia L, Chrysophyllum cainito, Piper marginatum, Lepianthes peltatum, Piper aduncum and Pimenta racemosa. The objective of this research was therefore to study the antimicrobial, cytotoxic and non-proliferation activities of the plants.
2. Method
2.1 â&#x20AC;&#x201D; Plant Species: Momoridica charantia L Momoridica charantia L. belongs to the family Cucurbitaceae. It is a flowering plant which is herbaceous. The plant, generally found in the tropics is commonly known as Cun de amor in the Dominican Republic. A tea preparation of the leaf is used for diabetes treatment of diabetes 2. A study showing its experimental use in mice showed improved glucose tolerance against Type II diabetes and a reduction in blood cholesterol3. Chrysophyllum cainito Chrysophyllum cainito, from the family Sapotaceae is native to the tropical America, from the Caribbean through Central
RESEARCH FEATURES America It’s a medium size tree that grows rapidly and reaches 20m in height. The plant is commonly known as Caimito in the Dominican Republic. Locally, the plant is used to treat diabetes. Piper marginatum Piper marginatum, member of the Piperaceae plant family is a perennial shrub. It’s native to the tropical America and the Caribbean. It has a heart-shaped leaves with 7-8 nerves, which ends at one point at the base. It has a strong smell. Just like other plants species in the Piperaceae family, this plant contains essential oils7. The locals use the plant as repellents, pain reliever, and fever treatment. Lepianthes peltatum Lepianthes peltatum, from the family Piperaceae is a light weight shrub of about 2m tall and 3cm in basal diameter. It’s commonly found in the tropical America and the Caribbean. It’s used for the treatment of fever and also used as pain killer. Previous study has suggested the presence on essential oil in the plant. This study will investigate its antimicrobial, antimitotic and cytotoxicity of the plant. Piper aduncum Piper aduncum, belonging to the Piperaceae plant family is a shrub up to 7m tall and 10cm or more in the stem diameter. It is native of the West Indies and the mainland tropical America. It’s locally known as Guayuyo in the Dominican Republic. The local people use it in the treatment of diabetes and kidney stones. Previous studies indicate the essential oil from the extract of the leaves had potential repellent activity against Ae. Aegypti, which is the main vector for Dengue fever7. Pimenta racemosa Pimenta racemosa, a plant in the family Myrtaceae is native to the La Espanola Island and is locally known as Ozua in the Dominican Republic. The tree is between 4-12m tall and the white flowers develop to become black, oval fruit between 7-12mm. In previous studies, the composition of the methanol extract has identified a pure compound known as Lupeol, a triterpene that has been found to have anti-phlogistic properties 3. The extract of this plant also contain some bioactive principles such as flavonoids and lactones1.
2.2 — Plant collection and extraction Six selected plants were collected and identified from sites in the Dominican Republic. The located sites included the vicinity of Punta Cana ecological resort and club and the city of Higuey. The leaves of the plants were allowed to dry in an open air for three days at a temperature of about 87 degree Fahrenheit. The dry plant leaves were then ground into
powder. The powdered plant leaves were dissolved in two different organic solvents, methanol and dichloromethanol at room temperature. The solvent from the mixture is called the crude extract. The resulting crude extracts were labeled as follows. Dichloromethanol extract (DCE) Momoridica charantia L (DCE 1) Chrysophyllum caniato (DCE 3) Piper marginatum (DCE 5) Lepianthes peltatum (DCE 7) Piper aduncum (DCE 9) Pimenta racemosa (DCE 11)
Methanol extract (ME) Momoridica charantia L (ME 2) Chrysophyllum caniato (ME 4) Piper marginatum (ME 6) Lepianthes peltatum (ME 8) Piper aduncum (ME 10) Pimenta racemosa (12)
2.3 — Use of Bioassay procedure A bioassay procedure is needed to find the bioactive properties of these plants. The crude extracts were used for discdiffusion assay, brine Shrimp assay and sea urchin embryo assay.
2.4 — Antimicrobial Assay (Disc-Diffusion Assay) Disc-Diffusion Assay was performed in order to investigate the antibacterial and antifungal functionalities of the crude extracts. Bacteria were collected from yellow dust spots on the leaves of a house plant, Syngonium podophyllum, using cotton swab. The yellow dust spots on the leaves is a common symptom of bacteria disease caused by Xanthomonas campestris. The bacteria were labeled PCI. Using cotton swab bacteria was collected from laboratory sink and labeled as PC II. Fungi were also collected by taking a scoop of the top layer of a moldy soil. Pathogenic fungi such as Exserohilum rostratum is commonly found in soil and plant mold4. The fungi was labeled as PC III. Filter paper Discs were dipped in crude extracts and were left to dry on aluminum foil. Each individual bacteria and fungi were spread over the petri dishes thoroughly to ensure even distribution of the microbes. Care was taken to prevent contamination. The organic solvents used for the extraction were set as the control and were applied the discs. The discs were then left to air-dry to remove all the solvent. Treatment filter paper disc were later placed on the petri dish containing the microbes with sufficient amount of space between them. The petri dishes were then incubated at 37 degree C. for 24 hours. Following incubation, the plates were inspected to identify the zone of growth inhibition.
2.5 – Brine-shrimp Assay-Cytotoxicity The Brine shrimp cytotoxicity assay was conducted in order to test the toxicity of the crude extracts to cells. Brine shrimp cells were used in this study because of its similarities to hu-
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RESEARCH FEATURES man cell11.The lethality of the crude extracts to the brimeshrimp cells will indicate potential toxicity to human cells. 1 ml solution of brine-shrimp cells were placed in six wells of a 5x4 well plate. The number of brine shrimps cells in each well was counted. 0.5ml of the methanol crude extract was later added to the first five wells. Methanol was used as the control in the last well. This process was replicated for 1ml and 1.5ml concentrations of the methanol crude extracts. The brine shrimp was recounted after 24 hours to find the number of brine shrimp that died. This process was repeated for 2 more trials and the percentage of average brine shrimp for each extract was computed.
2.6 - Sea Urchin embryo Assay- Antimitotic
3. Results
The Sea Urchin embryo assay was conducted in order to test the extracts for anti-proliferative activity. 2 mL of seawater were first spread into every well of a 5x4 well plate. Extracts were placed at concentrations of 0.5ml, 1ml or 1.5ml into their respectively labeled wells. Controls were run using methanol, and seawater. Sea urchins were gathered from the Punta Cana Resort boat dock in front of the Diving Center and placed on ice to prevent premature spawning. Spawning was induced in sea urchins by injecting approximately 0.5 mL of Potassium Chloride (KCl, 0.5N) through the soft tissue of the oral surface into the body cavity. Approximately 5 mL of eggs were collected inside of a beaker and were fertilized with around 200 ÂľL of sperm. One drop of fertilized egg solution was placed into each well.
3.1 - Antimicrobial Assay (Disc -Diffusion Assay) Methanol Extract (ME) ME 6 ME 8 ME 10 0 + +
Microbes ME 2 ME 4 ME 12 PCI +++ ++ ++ (Bacteria) PCII ++ + 0 ++ + ++ (Bacteria) PCIII +++ ++ + ++ + ++ (Fungus) Table 1. Antimicrobial (bacteria and fungus) effect of the methanol extracts.
Control 0 0 0
Dichloromethanol Extract (DCE) DCE5 DCE7 DCE9 0 0 +
Microbes DCE 1 DCE3 DCE 11 Control PCI ++ 0 +++ 0 (Bacteria) PCII + 0 0 0 + 0 0 (Bacteria) PCIII + + + + + + 0 (Fungus Table 2. Antimicrobial (bacteria and fungus) effect of the dichloromethanol extracts. Legend 0 No growth Inhibition (0mm) + Weak growth Inhibition (0-5mm) +++ Strong growth Inhibition (10mm or more) ++ Moderate growth Inhibition (610mm)
It was found that the methanol crude extracts showed significant growth of inhibition against the test microbes. Methanol crude extracts for Piper marginatum (ME 6) showed the weakest antimicrobial activity against the test microbes by showing no zone of growth inhibition against bacteria PCI and PCII. It also showed a very weak growth of inhibition against fungi PCIII. As seen from table 1, the rest of the methanol crude extracts showed modestly stronger zone of inhibition against the test microbes. Crude extract for Momoridica charantia !" !"##$%&"#%"'()*+#,-'.'/,--'0123
L (ME2) had the strongest antimicrobial activity against the test microbes by showing strong zone of growth inhibition against the bacteria PCI and fungi PCIII. It showed a moderate zone of growth inhibition against the bacteria PCII. It appears that methanol extract from Momoridica charantia (ME 2) showed strong inhibition against PC I and PC III where as a moderate inhibition was shown against PC II (Table 1). Methanol extract from Pimenta racemosa (ME 12) showed moderate inhibition against PC I, PC II and PC III. Piper marginatum
RESEARCH FEATURES
3.2 â&#x20AC;&#x201D; Brine Shrimp Assay (Cytotoxicity) Another purpose of this study was to investigate on the cytotoxic activity of the crude extracts on brine shrimp. As it appears, Momoridica charantia showed no toxicity against the brine shrimps upon addition of 0.5ml and 1ml of the extract but the 1.5ml of extract showed somewhat toxicity against the brine shrimps. (Figure 1). Chrysophyllum canito also showed no toxicity against the brine shrimps for one and two drops, but showed little toxicity for the 1.5ml of extract. Methanol extracts from Piper marginatum showed little toxicity against the brine shrimp for 0.5ml of extract and moderate toxicity for 1ml of the extract. However, it showed strong toxicity against the brine shrimps for 1.5ml of extract (Figure 1). Also, it was observed that Lepianthes peltatum showed little toxicity for 0.5ml and 1ml of extract and moderate toxicity for 1.5ml of extract. The Piper aduncum methanol extract showed moderate toxicity against the brine shrimps for 0.5ml whereas the 1ml and 1.5ml of the extract showed strong toxicity against the brine shrimps. For the Pimenta racemosa, no toxicity was shown against the brine shrimps for the 0.5ml, but little and moderate toxicity was seen against the brine shrimps for the 1ml and 1.5ml respectively.
3.3-Sea Urchin Assay â&#x20AC;&#x201D; Antimitotic
It was also noted that the dichloromethane crude extracts showed moderately weak growth of inhibition against the test microbes. From table two, the dichloromethane extract for momoridica charantia L show the strongest antimicrobial activity against the test microbes by showing moderate growth inhibition against the bacteria PCI and weak zone of growth inhibition against bacteria PCII and fungi PCIII. The rest of the dichloromethane as seen in table 2 showed weak antimicrobial activity against the test microbes. Dichloromethane extracts for Chrysophyllum cainito (DCE 3), Piper marginatum (DCE 5), Lepianthes peltatum (DCE 7) and Piper aduncum (DCE 9) showed weak or no inhibition (Table 2). Extracts from Momoridica charantia (DCE 1) showed moderate inhibition against PC I and weak inhibition against PC II and PC III. Pimenta racemosa (DCE 11) showed strong inhibition against PC I but no inhibition against PC II. A week inhibition of the DCE 11 was shown against PC III.
It was found that all the plant crude extracts showed significant non-proliferation activites. As it appears from figure 2, methanol crude extracts from piper marginatum (ME 6), lepianthes peltatum (ME 8), piper aduncum (ME 10) and pimenta racemosa (ME 12) showed very strong non proliferation activities. This discovery suggest the plantsâ&#x20AC;&#x2122; ability to stop cell division. As seen from figure 2, methanol crude extract from momoridica charantia (ME 2) showed moderate non-proliferation activity at 0.5ml whlie it showed very weak non-proliferation activity at 1.5ml. Crude extract from chrysophyllum canaito (ME 4) showed weak non-proliferation at 0.5ml and 1.5ml.
Figure 1: graph showing the cytotoxic activities of the plant extracts
(ME 6) and Piper aduncum (ME 10) showed weak or no inhibition against PC I, PC II and PC III. Furthermore, Chrysophyllum cainito (ME 4) and Lepianthes peltatum showed moderate inhibition against the microbes.
Figure 2: graph showing percent rate of non-proliferation activities of the plant crude extracts
4. Discussion & Conclusion One of the purposes of this study was to investigate the efficacy of six selected plants against microbes (Bacteria and fungi). In other to achieve such goal, an antimicrobial (disc- diffusion) assay was conducted. The strong zone of growth inhibition of the Momoridica charantia methanol extract against bacteria (Punta Cana II) sug-
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RESEARCH FEATURES gests antibacterial properties of this extract. Moderate growth inhibition of the crude extracts suggests antifungal activities of the plant. A previous study on this plant has shown that it is effective against diabetes3. According to the Johns Hopkins health alert, researchers have link the reduction of blood sugar level to certain phytochemicals present in this plant10.
preciation to Dr. Manuel Aregullin and Dr. Maria Laux who in anyway have contributed and inspired me to the overall success of this undertaking. My grateful thanks also go to all the visiting instructors of the 2011 MHIRT program at Cornell University. I am also grateful to my faculty adviser, Dr. Anne Osano who guided me through this endeavor.
Moderate zone of growth inhibition of the Pimenta racemosa may also suggest antimicrobial properties in the plant. Prior studies on the plant have suggested activities related to peripheral mechanism7.The actual mechanism and bioactive principles behind such action is still not well understood5. Further studies should investigate more information about this process. Methanol Crude extracts from Piper marginatum and Piper aduncum showed weak inhibition therefore suggesting little or no antimicrobial properties.
6. References
The strong cytotoxic activity shown in the Piper aduncum crude extract suggests anti-cell growth properties in the plant. Piper aduncum extract contain essential oils, which is also made up of dillapiole, a phenylpropanoid with reported insecticidal properties9. Extracts from the plant is used for the treatment of leishmaniasis, an infectious skin disease caused by protozoan parasite6. Moderate cytotoxic activity of Lepianthes peltatum suggests some kind of anti-cell growth properties in the plant. The non-proliferation activity of the crude extracts was also studied. The study was conducted by testing the extracts on the fertilized embryos of sea urchin. The aim of the study was to find out which extract can stop cell division by stopping the formation of the spindle fiber. The strong non-proliferation activities of the extracts from Piper marginatum, Piper aduncum and Lepianthes peltatum suggest anticancer activity of the plants. This finding is interesting given that all of these plants that showed such strong non-proliferation activity are from the Piperaceae plant family. One thing that is common in the Piperaceae plant family is the presence of essential oils1. Studies about these essential oils have suggested their anti-inflammatory properties. Although this study did not further investigate on the constituent of those plants that may cause such property, it may be reasonable to infer that the essential oil content in the plants may have a contributory factor for their non-proliferation activites. Many questions come to mind in terms of what chemical and biological aspect of the plants may cause such potency in against cell proliferation. Very little has been explained about the biochemical mechanisms in this research thus far.
5. Acknowledgements I wish to express my deepest gratitude and warmest ap!" !"##$%&"#%"'()*+#,-'.'/,--'0123
1. Alvarez, A. et al. (2004). Antibacterial activity of essentialoil of pimenta racemosa var. terebinthina and pimenta racemosa var. grisea.fitoterapia . 2. Ayensu ES, DeFilipps RA. Endangered and Threatened Plants of the United States. Washington, DC:Smithsonian Institution, 1978. 3. Bakare , R. I. et al (2010). Nutritional and chemical evaluation of momoridica charantia. Journal of medicinal plants research, 4(21), 2189-2193. 4. Centers for Disease Control and Prevention, (2012). Fungal diseases. Retrieved from website: http://www.cdc.gov/fungal/ other/exserohilum.html 5. Fabricant D.S and Farnsworth N.R, 2001. The value of plants used in traditional medicine for drug discovery. Environ Health Perspect. 2001 March; 109(Suppl 1): 69â&#x20AC;&#x201C;75.PMCID: PMC1240543 6. Fernandez, M. A. et al (2001). Anti- inflammatory activity of abietic acid, a diterpene isolated frompimenta racemosa var. grissea. Journal of Pharmacy and Pharmacology, 7. Garcia , M. D. et al. (2004). Antinoceptive and anti-inflammatory effect of the aqueous extract from leaves of pimenta racemosa var. ozua (mirtaceae) . Journal Of Ethnopharmacology, 8. Martinez, J. et al (2011). Extraction of volatile oil from piper aduncum l. leaves with supercritical carbondioxide. 9. Misni, N. et al. (2008). The repellent activity of piper aduncum linn (family:piperaceae) essential oil against aedes aegypti using human volunteers . The Journal of Tropical medicine and parasitology, 31(2). 10. Parker, M. I. (2010). Domestication syndrome in caimito (chrysophyllum cainito l.): Fruit and seed characteristics. Economic Botany, 64(2), 161-175. 11. Silva, W. C. (2009). Veterinary parasitology 12.Verpoorte R. Pharmacognosy in the new millennium: leadfinding and biotechnology. J Pharm Pharmacol 52:253â&#x20AC;&#x201C;262 (2000).
RESEARCH FEATURES
Elucidating RNA Silencing Pathways by Identifying Mutant Knockouts within Arabidopsis Thaliana Michael Schatz, University of Pennsylvania Research Advisor: Dr. Brian Gregory, Department of Biology MicroRNA and small interfering RNA play an essential role in the regulation of gene expression. By binding to complementary messenger RNA, these small RNA are capable of inducing RNA inhibitory pathways. Dicer, dicer-like (dcl) proteins, and DAWDLE (DDL) proteins are believed to be involved in the production of microRNA and its precursors, whereas argonaute (Ago) proteins have been implicated in the stabilization of the RNA-induced silencing complex—a structure crucial for providing microRNA with the right orientation to bind messenger RNA. RNA-dependent RNA polymerases (RDRs) also contribute to RNA silencing pathways by producing double stranded RNA—an important precursor to micro and small interfering RNA. Since all of these proteins are essential for the biogenesis of small RNAs, a mutation in the genes encoding them will undoubtedly result in changes to RNA silencing pathways. The aim of this project was to identify strains of Arabidopsis Thaliana with knockout mutations in these genes so that they may eventually be used as a tool in the lab to explore the functional role and target molecules of the proteins they encode. Within Arabidopsis, we have identified single homozygous knockouts for the RDR 4 and possibly DDL genes, a double homozygous knockout for RDR 2 and RDR 6, and heterozygous mutants for both the ago1/+ and vsc6 het genes. We were not able to isolate homozygous knockouts for the RDR 3 gene or heterozygote mutants for the dcl1-7/+ genes.
I. Introduction In some ways, regulation of gene expression is as important to an organism as the DNA it inherits. Differential gene expression is responsible for developmental cues within organisms, cellular responses to changing external stimuli and cellular specialization. It is also essential to understanding the treatment and/or propagation of diseases. Gene expression can be regulated at virtually any part of the pathway from DNA to RNA: histone modifications and chromosomal packing, transcriptional interference, transportational obstacles, translational interference, post-translational degradation, and so on. It is now known that many of these regulatory processes are mediated directly by ribonucleic acids (RNA) in addition to proteins. The known scope of RNA function has exploded in recent years well beyond its traditional informational role in protein coding. It is now even believed that it is the ribosomal RNA (rRNA), and not the ribosomal proteins, that provide the catalytic site in ribosomes7. MicroRNA (miRNA) and its synthetic analog, small interfering RNA (siRNA), contribute to RNA interfering (RNAi) pathways by binding to messenger RNA (mRNA). Precursor miRNA is transcribed by an RNA polymerase, and then capped and polyadenylated1. Due to the self-complementarity of the precursor miRNA, it forms a hairpin structure that is subsequently processed and cut into a microprocessor complex by the Drosha enzyme3. It is believed that Dicer proteins cleave the double stranded RNA of the miRNA precursor into a fragment of approximately 25 nucleotides5. Argonaute (Ago) proteins within the RNA induced silencing
(RISC) complex then help orient the miRNA strand so that it can bind mRNA. According to Lim et al., perfect complementarity between the miRNA and mRNA can allow Ago proteins to cleave the mRNA whereas evidence shows that partial complementarity only achieves gene regulation by occluding translation on the ribosome rather than inducing the degradation of mRNA4. One benefit in studying these sequence dependent silencing mechanisms is that if one knows the sequence, the target can be deduced. Double-stranded RNA (dsRNA) is crtical for the production of miRNA, which in turn is a pivotal part of the RISC complex and RNAi pathway. As described above, cells can make dsRNA by using RNA polymerases to transcribe fragments of DNA with self-complementary transcripts and by subsequently cleaving and processing the resulting hairpin structure with accessory proteins. Alternatively, RNA-dependent RNA polymerases (RDRs) within the cell can use single stranded RNA as a template to help synthesize a complementary strand, and thereby produce dsRNA. One way to study RNAi pathways is to analyze the effects of mutations to genes encoding various proteins believed to be part of the regulatory process. We studied mutations within the model organism Arabidopsis Thaliana. Arabidopsis has the advantage of a short generation time and extensive previous research. An important tool for the generation of Arabidopsis mutants is the bacteria Agrobacterium which is capable of inducing tumor-like growths by using plasmids to semi-randomly insert transfer DNA (T-DNA) segments into the host cell’s genome2. By disrupting the gene in which
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RESEARCH FEATURES it inserts, these T-DNA segments disturb gene function. Agrobacterium can be engineered so that instead of causing tumors, they contain a selectable marker such as herbicide resistance. Large consortiums like the SALK institute have compiled extensive libraries with strains of Arabidopsis with T-DNA inserts in particular gene locations. We explored Arabidopsis strains with T-DNA inserts in the genes encoding RNA-dependent RNA polymerases, argonautes, dicer-like (DCL), and DAWDLE (DDL) proteins. DAWDLE is a protein with a forkhead-associated domain that is believed to aid the production of miRNA within Arabidopsis by interacting with the Drosha enzyme and helping DCL1 proteins identify precursor miRNA molecules10. Since each of these genes code for proteins that have essential roles in the production of dsRNA and RISC complex, their knockouts will lead to up-regulation of their target genes. In particular, by comparing knockout mutants to wild types one can gain an understanding of the role these genes have in regulating gene expression. Before potential target genes can be deduced, however, it is first necessary to identify the knockouts. Our goal was to identify single homozygous knockouts for RDR 2, RDR 3, RDR 4 and DDL genes, a double homozygous knockout for RDR 2 – RDR 6 genes, as well as heterozygotes with mutations in Ago1 or dcl1-7 genes, within Arabidopsis Thaliana.
2. Experimental Procedure Strains Strains of Arabidopsis Thaliana used in this report were received from the University of Pennsylvania, Salk Institute and SAIL consortium. Soil Preparation and Planting Diatomaceous earth was added and mixed into Fafard soil in order to prevent invasion by any potential insect larvae. A mask was worn during this process in order to avoid inhalation of airborne diatoms. This soil was then added to clean flats. Since the large amounts of perlite in the soil make it more difficult to identify herbicide resistant mutants during the harvesting phase, the soil and flat are tapped gently and the excess perlite that rises to the top is removed. The soil was then watered from bottom up and top down. The tops of the soil were also doused with a 20-20-20 fertilizer mixture of a concentration of approximately 1 teaspoon per 1.5 gallon of water, or ¼ teaspoon per flat8. Arabidopsis seeds were then added to all labeled wells within the flat. In order to aid the identification of the herbicide resistant mutants during the harvesting phase, seeds were evenly distributed in each well rather than concentrated at one spot. The flats were then placed in an incubation chamber and allowed to grow for four weeks before harvesting. As the plants grow, !" !"##$%&"#%"'()*+#,-'.'/,--'0123
they are sprayed with herbicide. Wells with wild type, herbicide-sensitive plants, as indicated by dead leaves or leaves with brown or white discolorations, were removed from the flat in order to prevent potential cross spread into wells with mutant plants. It is easiest to identify herbicide resistant mutants before the plants fully mature and for this reason it is recommended to screen the plants well before the end of its four-week incubation. DNA Isolation Using ethanol-sterilized forceps, green leaves were individually harvested from plants in each well and added to labeled 1.5 mL microcentrifuge tubes. The microcentrifuge tubes with plant leaves were then chilled in liquid nitrogen or dry ice. While chilled, the plant tissue was pulverized in its respective microcentrifuge tube using fitted autoclaved pestles. 150 µL of 2X CTAB was added to each sample, and each sample was further ground up using the pestles. At this point the mixture appeared homogenous with little or no leaf chunks. The samples were incubated in a hotplate at 65˚C for at least 10 minutes and no more than two hours. After letting the samples cool at rest for a few minutes, 150 µL of chloroform was added to each and mixed in using a vortex machine. The samples were then microfuged at max speed for 15 minutes to separate the aqueous and organic phases. The aqueous layer of each respective sample was pipetted into a microcentrifuge tube with 150 µL of isopropanol. The samples were again mixed by vortex and then spun at max speed for 5 minutes to allow for DNA pellet formation. After decanting the isopropanol, each DNA pellet was washed with 250 µL of 70% ethanol and then microfuged at maximum speed for another 5 minutes. The ethanol was then carefully pipetted out from each sample. After allowing residual ethanol to evaporate overnight, each DNA pellet was finally dissolved and suspended in 50 µL of TE buffer6. RNA Extraction Unopened flower buds of select plants were harvested using ethanol-sterilized forceps and placed directly into respectively labeled, liquid nitrogen chilled microcentrifuge tubes. While chilled in liquid nitrogen, the flower buds from each sample were pulverized using fitted autoclaved pestles. An adapted QIAGEN kit and protocol was then used to extract RNA from the samples. The RNA samples then underwent reverse transcription polymerase chain reaction (RT-PCR) and the subsequent products were analyzed on an ethidium bromide stained 4% agarose gel. DNA Amplification Before DNA amplification, the DNA samples suspended in TE were vortexed and briefly centrifuged at maximum speed in order to ensure a homogenous solution. Their concentrations were assessed using a NanoDrop spectrophotometer
RESEARCH FEATURES and each sample was subsequently diluted to 50 ng/µL.
mozygous or heterozygous mutation.
PCR plates contained 15 µL of sample per well: 2 µL of DNA sample and 13 µL of the master mix solution. The master mix solution was composed of nuclease free water, 10x standard taq buffer, 10 mM dNTPs, forward primers, reverse primers, taq polymerase in ratio of 9.5: 1.5: 0.5: 0.5: 0.5: 0.5 by volume.
3. Results
Restriction Digest In order to ensure same-lengthed DNA fragments, some samples were put through a restriction digest. After adding the restriction digest, the samples were amplified through PCR. Product Visualization and Analysis Amplified DNA was analyzed on 1 or 4% ethidium bromide stained agarose gels. 15 µL of each amplified DNA along with about 1 µL of loading dye was pipetted into its own well with the gene specific and insert specific amplified DNA usually staggered with each other in order to facilitate analysis. Mutant DNA was run alongside 1 Kb+ DNA ladder, wild type DNA and a few no-template-controls (NTC). Since the NTC contained 13 µL of master mix solution but no DNA sample, they were useful in identifying potential contamination. Gels were run for approximately 20 to 30 minutes between 100 and 160 volts. Gel images were captured under UV light. Two different methods were employed to amplify the DNA. In the insert specific PCR, a pair of primers are chosen such that one will border a sequence within the T-DNA insert, and the other will border a sequence within the gene of interest. Since only the mutants have the T-DNA insert, the PCR will only amplify heterozygous or homozygous mutants (non-mutated DNA will not be amplified). In the gene specific PCR a pair of primers are chosen so that each borders a sequence on the gene of interest. The PCR conditions are chosen so that if there is a T-DNA insert between the two primers, then the sequence is too long and will not be amplified. The elongation time, however, is just right for the wild type gene and therefore the PCR will only amplify the normal gene and not the mutant. In this way gene specific PCR identifies heterozygote and homozygote wild types. Combining these two methods is a powerful technique that allows for the delineation of homozygote wild types, and homozygote and heterozygote mutants. Results were analyzed according to the theory that homozygous wild types should amplify DNA with gene specific primers and not insert specific primers, whereas homozygous mutants with the insert mutations should only amplify the DNA with insert specific primers; finally, heterozygotes should amplify both. By comparing the gel bands of the various samples with those of the wild type it was possible to determine whether or not a particular sample harbored a ho-
RNA-Dependent RNA Polymerases:
Identify homozygous knockout mutants. RDR 3: 43 samples of SALK071908 and 31 samples of SALK137401 were genotyped and none of them yielded amplification of the insert PCR product, therefore no RDR 3 homozygote knockout mutants were identified. RDR 4: 49 samples of SALK143709 were genotyped with nearly all of them showing positive insert specific bands. There were some faint bands in the gene specific lanes as well, but they did not migrate quite as far as the gene specific bands in the wild types (Figure 1). After reverse transcription PCR, one sample showed no RDR 4 transcript (Figure 2), thereby confirming at least one RDR 4 homozygous mutant knockout. Figure 1: Gene Specific and Insert Specific Gel for Potential RDR 4 Mutants
Note: Gene specific PCR products are run alongside the insert PCR product, with the gene specific product on the left of each respective labeled column Figure 2: RT-PCR Products of Selected Potential RDR Mutants
RDR 2X6: Homozygote RDR 2 knockouts were crossed with homozygote RDR 6 knockouts and 38 samples of the resulting generation were genotyped at the RDR 2 locus. There were some gene specific bands for the samples but they were at slightly different locations compared to the
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RESEARCH FEATURES wild types (Figure 3). Selected samples were then tested for insert specific PCR products. Two different insert primer pairs were used (Figure 4). Five of the selected samples showed positive insert specific bands for both primer pairs thereby strongly indicating homozygous mutant knockouts. However, when DNA from these 5 samples was re-isolated in order to provide a confirmation of the previous results, only 2 confirmed the previous results. Selected samples were then genotyped at the RDR 6 locus and the results indicated that most were homozygous mutants. In conclusion, at least 2 samples of RDR 2, RDR 6 double mutants were identified. Argonaute: Since homozygote knockout mutations in the Argonaute genes are sterile, we looked for heterozygotes in order to propagate the line. Ago1/+: 17 samples of SALK089073 and 20 samples of SALK076191 were genotyped. 9 samples of SALK089073 showed amplification for both the gene specific and insert specific PCR products, thereby indicating potential heterozygote mutants (Figure 5). Re-isolation of selected SALK089073 samples confirmed the presence of heterozygote mutants. SALK076191 samples showed no insert specific bands and therefore no heterozygotes could be
identified from that strain. Seg ago1 exon and severe ago1 exon: 20 samples of SALK 119341 and 21 samples of SALK 116845 were genotyped. Both strains showed some amplification for the gene specific PCR products but not the insert specific PCR products (Figure 6). Both strains were tested again with different insert primer pairs but none really showed any noticeable unique amplification.
Dicer-like:
As with the Argonaute genes, homozygous knockout mutants for the dcl1-7/+ gene are sterile and therefore we sought to identify heterozygotes for propagation of the line. Dcl1-7/+: 8 samples of SP7202-2 and 11 samples of SP7202-4 were genotyped. After running a PCR on these samples, Stu1 was used to do a restriction digest. The resulting products were imaged on a 4% agarose gel. Numerous complicating factorsâ&#x20AC;&#x201D;such as poor gel resolution and omission of a positive control for the sp72024 lineâ&#x20AC;&#x201D;contributed to the inconclusive results (Figure 7). In the future, these samples should be run with all the positive controls, and perhaps under a different annealing temperature.
Figure 3: Gene Specific PCR Products at RDR 2 locus for Potential RDR 2x6 Mutants
Figure 4: Insert Specific PCR Products at RDR 2 locus for Potential RDR 2x6 Mutants
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RESEARCH FEATURES Figure 5: Gene Specific and Insert Specific Gel for Potential Heterozygote Ago1/+ mutants
Note: The left lane of each respective twolane column is the gene specific PCR products and the right column is the insert specific PCR products
Figure 6: Gene Specific and Insert Specific Gel for Potential Heterozygote Seg Ago1 or Severe Ago1 Mutants Note: The left lane of each respective twolane column is the gene specific PCR products and the right column is the insert specific PCR products
Figure 7: Image of SP7202-2 and SP702-4 samples Post-Restriction Digest
DAWDLE: Identify homozygous mutant knockouts. DDL: 13 samples of sail 1281-FO8 were genotyped. Several samples showed no gene specific band but the insert specific primer pair did not work (Figure 8). Consequently, we were only able to identify possible homozygous knockout mutants. Future experiments should test different insert specific primer pairs on these samples in order to confirm whether or not they are really homozygous mutants. DDL_97 144: 21 samples of CS85849 were genotyped and subsequently digested with HIN 4I. There were a few po-
tential homozygotes but the image resolution made it difficult to determine anything for sure (Figure 9). In the future, when this experiment is repeated, the post-restriction digest projects should be run on the gel for a longer time in order to get better band separation and resolution. Varicose: Identify heterozygous mutants vcs6 het: 24 samples of vcs6 het were genotyped. There was amplification in both the gene specific and insert specific PCR products therefore giving strong evidence of heterozygous mutants (Figure 10).
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RESEARCH FEATURES Figure 8: Gene Specific and Insert Specific Gel for Potential Homozygous DDL Knockout Mutants
Note: The left lane of each respective two-lane column is the gene specific PCR products and the right column is the insert specific PCR products Figure 9: Image of CS85849 Samples Post-Restriction Digest
Figure 10: Gene Specific and Insert Specific Gel for Potential vsc6 het Heterozygous Mutants
4. Discussion The goal of this project was to identify strains of Arabidopsis with mutations in genes encoding RNA-dependent RNA polymerases, DAWDLE proteins, argonaute proteins and dicer-like proteins. We have successfully isolated single homozygote mutants for RDR 4 and a double homozygous mutant for RDR 2x6, possible homozygous mutants for DDL and heterozygous mutants for both ago1/+ and vcs6 het. These identified mutants can now be used as tools to help deduce the functional role and scope of these proteins and the targets they may affect. Though we were able to identify many homozygous and heterozygous mutants, some of the strains proved difficult. Possible reasons for failure in these strains include, but are not limited to absence of T-DNA insertion, T!" !"##$%&"#%"'()*+#,-'.'/,--'0123
DNA insertion into a different gene locus, or choice of wrong primer pairs. In any of these cases there would be no amplification of the insertion specific PCR products. Throughout the project it was usually the insert specific, and not gene specific, primer pairs the posed the most problems. Furthermore, it is possible that some of the mutants have a phenotype that made it predisposed to dying at an early stage. If this is the case, then during the harvesting phase, a disproportionately high amount of wild type tissue will be harvested. There were also potential issues with contamination. In order to confirm the presence of homozygous knockouts at the RDR 2 gene locus of the RDR 2, RDR 6 double mutant, DNA from selected samples were re-harvested. When their PCR products were analyzed, however, some of the samples were not confirmed. A possible explanation for this is cross contamination by plants adjacent to the sample in the flat. Another possibility is that the
RESEARCH FEATURES quality of the second DNA extraction wasn’t as good as the first. This could be because the plant tissue was older. Lastly, on some of the gels the no template controls had bands. Since no template controls are designed to not have any DNA sample in them, this finding is highly suggestive of contamination. The next step in this process is to quantitatively compare the single stranded and double stranded RNA of mutant knockouts with that of the wild type. This can be done through high-throughput sequencing. Genes whose knockout results in lowered levels of a particular dsRNA may be presumed to be involved in its regulation. In this fashion the targets of the normal functioning gene can be deduced. Likewise, up-regulation of the complementary single stranded RNA within the mutant knockouts would further confirm the identified targets and suggest functional roles. Presumably some of these proteins work synergistically to bring about RNA inhibition. It is for this reason that we sought to identify a double homozygous knockout mutant for both RDR 2 and RDR 6. The single stranded and double stranded RNA within this double mutant can be compared with that in the respective single stranded knockouts. Differences between the double stranded and single stranded profiles can then be used to deduce the effects of interactions between these two RDRs. Theoretically this principle can be extended to triple homozygous mutant knockouts and multiple mutations in different genes, e.g. the RDR 2 and Ago1 gene, or perhaps the RDR 6, dcl1-7 and Ago1, etc. This method allows us to not only identify the targets and functional roles of these genes, but also deduce the complex ways in which their proteins interact with each other.
5. Author Contributions All experiments outlined in this report were carried out with equal collaboration from Sager Gosai. In addition to helping identify herbicide resistant mutants, Matthew Willmann also helped with some of the soil preparation and seed planting.
ing me to the DNA extraction protocol. I would like to especially acknowledge Dr. Matthew Willmann for introducing me to every other protocol and for his guidance throughout the project. Lastly, I would like to thank Dr. Brian Gregory for constantly reminding me that I can go wherever biology takes me. Without his vision this project would not have been possible.
7. References 1. Cai X, Hagedorn CH, Cullen BR (December 2004). Human microRNAs are processed from capped, polyadenylated transcripts that can also function as mRNAs. RNA 10 (12): 1957– 66. doi:10.1261/rna.7135204. PMC 1370684.PMID 15525708. 2. Francis, K. E.; Spiker, S. (2004). Identification of Arabidopsis thaliana transformants without selection reveals a high occurrence of silenced T-DNA integrations. The Plant Journal 41 (3): 464–477. doi:10.1111/j.1365-313X.2004.02312.x.PMID 15659104. 3. Gregory RI, Chendrimada TP, Shiekhattar R (2006). MicroRNA biogenesis: isolation and characterization of the microprocessor complex. Methods Mol. Biol. 342: 33–47. doi:10.1385/1-59745-123-1:33. ISBN 1-59745-123-1. PMID 16957365. 4. Lim LP, Lau NC, Garrett-Engele P, Grimson A, Schelter JM, Castle J, Bartel DP, Linsley PS, Johnson JM (February 2005). Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature 433 (7027): 769– 73. Bibcode:2005Natur.433..769L.doi:10.1038/nature03315. PMID 15685193. 5. Macrae I, Zhou K, Li F, Repic A, Brooks A, Cande W, Adams P, Doudna J (2006). Structural basis for double-stranded RNA processing by Dicer. Science 311: 195-198. 6. Murray MG, Thompson WF (1980) Rapid isolation of highmolecular-weight plant DNA. Nucl Acid Res 8: 4321-4325 7. Rodnina MV, Beringer M, Wintermeyer W (2007). How ribosomes make peptide bonds. Trends Biochem. Sci.32 (1): 20–6. doi:10.1016/j.tibs.2006.11.007.PMID 17157507. 8. Willmann, M. (2011). How to grow Arabidopsis plants. Gregory Lab, UPenn.
6. Acknowledgements Thanks to the SALK institute, SAIL consortium, and University of Pennsylvania for providing the Arabidopsis strains. I would like to acknowledge Sager Gosai for collaborating with me on this project and for his delightful deadpan humor which helped lift spirits during long days. I would also like to thank Sara Foster for introduc-
9. Willmann, M. (2013). TAIL PCR Protocol. Gregory Lab, UPenn. 10. Yu B, Bi L, Zheng B, Ji L, Chevalier D, Agarwal M, Ramachandran V, Li W, Lagrange T, Walker JC, Chen X. (2008). The FHA domain proteins DAWDLE in Arabidopsis and SNIP1 in humans act in small RNA biogenesis. Proc Natl Acad Sci 105(29): 10073-10078. doi: 10.1073/pnas.0804218105
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